LIBRARY 


UNIVERSITY  OF  CALIFORNIA. 


Gl  FT    OF 


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Japanese  Lac-  -Ki-urushi 


ALVI3O   B     9TEVENS 


Contribution  to  the  Knowledge  of 
Japanese  Lac        ' 

(Ki-urushi) 


THESIS 

Presented  to  the  Philosophical  Faculty  of  the  University  of 
Bern,  for  the  Degree  of  Doctor  of  Philosophy. 


By  ALVISO  B.  STEVENS 

University  ol  Michigan 
Ann  Arbor,  Michigan,  United  States  ol  America 


ANN  ARBOR: 

The  Ann  Arbor  Pitts,  Printers 
1906 


JAPANESE  LAC  (KI-URUSHI) 


Doubtless  nearly  every  one  has  seen  and  admired  the  beau- 
tiful Japanese  vases  or  boxes  without  realizing  that  they  were 
finished  with  the  most  indestructible  varnish  known  to  man. 
There  are  at  present  vases,  more  than  a  century  old,  that  l^ave 
retained  their  beautiful  luster  so  perfectly  that  they  look  as 
though  they  had  been  finished  but  yesterday.  The  hardened 
surface  formed  by  the  genuine  Japanese  lac,  is  practically  un- 
affected by  the  usual  reagents,  which  are  so  detrimental  to  most 
varnished  surfaces,  as,  alcohol,  ether,  alkalies  and  acids.  It  is 
acted  upon  to  some  extent  by  strong  sulphuric  or  nitric  acids, 
and  may  be  dissolved  by  continued  heating  in  fuming  nitric  acid. 

Rein1  states  that  the  Japanese  doubtless  received  their  knowl- 
edge of  the  lac  industry  from  the  Chinese  in  the  early  part  of  the 
third  century ;  but  that  its  use  did  not  attain  great  importance 
before  the  middle  of  the  seventh  century.  Kotoku-Tenno,  the 
36  Mikado  (645  to  654  A.  D.)  had  a  ceremonial  head  covering 
of  paper,  which  was  covered  with  black  lacquer.  There  is  a 
lacquered  scarf  box  in  the  temple  at  Nara,  which  belonged  to 
a  priest  in  the  time  of  Kinnari  Tenno  (540  to  572  A.  D.). 

For  centuries  its  use  and  production  remained  a  secret.  As 
late  as  1873  wc  ^nc^  tne  statement  that  "The  manner  of  prepar- 
ing the  varnish  and  the  mode  of  applying  it,  is  likely  to  remain 
a  secret."2  In  the  following  year  Prof.  J.  J.  Rein  made  a  thor- 


1  J.  J.  Rein's  The  Industries  of  Japan,  London,  1889,  Lacquer  Work, 
PP-  339-377;  Rein,  Japan  II,  Leipzig  1886.     This  Author  has  minutely  de- 
scribed the  lac  industry  and  it  is  to  his  excellent  work  that  I  shall  fre- 
quently refer. 

2  Belfour's  Cyclopaedia  of  India. 


ough  study  of  the  method  of  collecting  and  applying  the  lac. 
He  describes  the  tree,  Rhus  vernicifera,  from  which  the  lac  is 
obtained,  as  follows: 

' 'Lacquer  trees  grow  up  straight  and  have  fairly  symmetrical 
crowns.  The  young  trees  have  fine,  large,  pinnate  leaves,  which 
in  good  soil  often  grow  to  be  more  than  a  meter  long,  and  far 
exceed  all  other  species  of  Rhus  in  size  and  beauty.  The  leaves 
are  unequally  pinnate  and  have  long  stems.  Before  falling  off 
in  October  they  become  yellow  or  reddish  brown.  There  are 
from  nine  to  fifteen  leaflets,  large,  oval,  pointed  and  unindented, 
which  have  fine  short  hairs  on  the  under  side. 

"In  June  appear  loose,  greenish  yellow  branches  of  blossoms, 
from  numerous  axils  near  the  end  of  the  thick  twigs.  The  fruit 
is  ripe  in  the  second  half  of  October,  is  yellowish  green  and  re- 
mains hanging  all  winter,  though  usually  gathered  in  November. 

"The  two  sexes  are  separate.  Therefore  when  the  chief  ob- 
ject of  its  cultivation  is  the  manufacture  of  wax  from  the  seed, 
the  male  trees  should  be  avoided,  reproduction  being  obtained  by 
root  sprouts  from  female  specimens.  The  trees  begin  to  bear 
fruit  when  eight  years  old  and  increase  in  productiveness  until 
thirty  or  forty  years  old." 

Shirasawa  gives  the  following:3  Rhus  vernicifera,  D.  C., 
Syn.  Jap.  Urushi-no-ki,  Fam.  Anacardiaceae. 

"Cultivated  in  the  countries  of  the  temperate  zone,  i.  e.  Shin- 
ono,  Kai,  etc.,  and  the  provinces  to  the  northeast  of  Honshiu. 
It  does  not  grow  in  warm  regions,  and  thrives  best  in  moist  soil. 
The  tree  attains  its  growth  quickly  and  reaches  a  hight  of  10 
meters,  and  the  trunk  a  diameter  of  four  decimeters. 

"The  buds  are  of  a  pyramidal  form,  short  with  curved  points 
and  covered  with  hairs  of  a  brown,  ash-gray  color  and  glistening ; 
cicatrix  of  the  leaves  large,  heart  shaped ;  pith  large. 

"Flowers,  end  of  May ;  fruit,  end  of  October. 

"The  wood  is  soft  and  brittle,  with  a  remarkable  difference 
in  the  color  between  the  sap  wood,  which  is  white  and  the  heart 


3  Iconographie   des   Essences   forestieres   du   Japan,     par,   M.    Homi 
Shirasawa,  1899,  P-  94- 


wood  which  is  yellow.     Air  dried  0.51   sp.  gr.,  kiln  dried  0.45 
sp.  gr. 

"The  wood  is  used  for  the  manufacture  of  utensils,  furni- 
ture, bric-a-brac,  wood  engraving,  etc. 

"The  lac  is  gathered  in  China,  of  which  the  provinces  Noto, 
Iwashivo  are  renowned.  Wax  is  collected  from  the  fruit." 

Doubtless  the  principal  source  of  vegetable  wax  is  Rhus 
succedanea  and  R.  sylvestris.  The  former  is  cultivated  exten- 
sively in  the  warmer  parts  of  Japan,  south  of  latitude  35°  N. 
Japan  but  is  cultivated  principally  between  latitudes  35°  and  39°, 

The  lacquer-tree  grows  in  nearly  all  parts  of  China  and 
including  the  provinces  of  Etschizin,  Schmano,  Aidzo,  Yoshino 
and  Yamato.  The  greatest  yield  is  from  trees  15  to  20  years  old, 
but  the  age  of  the  trees  when  the  lac  is  collected  varies  in  different 
localities,  in  some  places  at  from  five  to  six  years  old  when  the 
stem  is  the  size  of  a  man's  arm  and  in  other  localities  at  from 
nine  to  ten  years  old.  The  time  of  collecting  is  from  April  or 
May  to  the  first  of  November. 

The  tree  and  its  anatomic  relations  are  given  in  detail  by 
Moebius*.  He  states  that  schizogenic  lacticiferous  canals  are 
present  in  all  parts  of  the  plant.  These  contain  the  milk  juice 
that  exudes  after  incision. 

METHOD  OF  COLLECTING  THE   LAC,   AND   ITS   PROPERTIES.* 

The  peasants  sell  the  trees  to  the  lac  merchants  who  employ 
collectors  to  gather  the  lac.  Each  workman  operates  upon  from 
600  to  800  old  trees,  or  1000  young  trees  in  a  season.  He  begins  at 
the  bottom  of  the  tree  and  makes  horizontal  incisions  through  the 


*  D'er  Japanishe  Lacbaum,  Rhus  vernicifera  D.  C.  Eine  Morphio- 
logishe  anatomische  Studie  aphandlungen  der  senkenbergischen  naturfor- 
schenden  Gasellschaft,  Band  XX,  Heft  II. 

5  The  principal  facts  have  been  tv.ken  from  Rein's  Industries  of 
Japan,  but  reference  is  also  made  to  "JaPamscner  Lack"  by  Dr.  Wagner, 
Dingler's  polyt.  Journal,  218,  1875,  pp.  361-367. 

The  above  description  is  nearly  the  same  as  that  given  by  Ishimatsu, 
which  he  states  was  partly  taken  from  an  account  of  the  urushi  manufac- 
ture published  for  the  use  of  the  Japanese  primary  schools.  Manchester 
Literary  and  Philosophical  Soc.  3  series,  7,  1882,  p.  449. 


—  8  — 

bark  about  six  millimeters  wide8  with  the  sharp  side  of  the  Kaki- 
gaina,  a  hook  shaped  instrument  (No.  10,  p.  10).  He  then  repeats 
the  operation  on  the  other  side  of  the  tree  about  15  or  20  c.m. 
higher.  Thus  alternating  from  side  to  side  until  he  has  cut  as 
high  as  he  can  reach,  making  from  six  to  ten  grooves  on  a  side, 
which  extend  about  half  way  round  the  tree.  He  then  goes  to 
other  trees  until  about  ten  or  fifteen  trees  have  been  cut  after 
which  he  returns  to  the  first  trees  and  collects  the  raw  lac.  This 
is  removed  with  the  Natsu-bera,  an  iron  spatula  with  a  curved 
point  (No.  II,  p.  10),  and  scraped  into  the  Go,  a  small  wooden  or 
bamboo  pail,  which  the  workman  carries  in  his  left  hand.  After 
about  four  days  he  returns  to  the  first  group  of  trees  and  cuts 
grooves  parallel  to  each  of  the  first.  These  operations  are  re- 
peated at  intervals  until  the  tree  is  literally  covered  with  grooves. 
The  entire  operation  requires  from  60  to  100  days. 

The  juice  usually  fills,  but  does  not  flow  out  of  the  grooves. 
In  the  spring  the  lac  is  thin,  gradually  becoming  thicker  as  the 
season  advances.  The  best  is  collected  in  mid-summer.  When 
collected  the  juice  is  in  the  form  of  a  thick  grayish- white  emul- 
sion, which  on  exposure  to  air  rapidly  changes  to  brown  and 
finally  to  black.  If  left  in  an  open  can  it  rapidly  forms  a  black 
skin  over  the  surface  which  prevents  further  oxidation.  The 
lac  collected  as  above  is  the  best  quality,  known  as  "Ki-Urushi" 
and  has  a  sp.  gr.  of  1.002  to  1.0379.  When  strongly  magnified 
it  appears  to  be  a  brownish  mass  of  globules,  which  consists  of 
two  kinds,  one  kind  small,  dark  brown  and  between  these  a  less 
number  of  large,  light-colored  globules,  the  former  soluble  in 
alcohol  and  the  latter  in  water.  A  second  grade  known  -as 
"Seshime-Urushi"  is  obtained  at  the  close  of  the  season  by  cut- 
ting down  some  of  the  trees  and  cutting  and  binding  the  branches 
into  bundles  about  one  metre  long  and  these,  with  the  trunk  are 
macerated  in  warm  water  when  the  sap  comes  to  the  surface  and 
is  removed.  Wagner7  states  that  after  maceration  the  branches 
are  placed  in  a  screw  press  to  remove  the  juice.  This  is  thin  and 
dark  and,  after  mixing  with  some  drying  oil,  is  used  as  an  under 
varnish.  Each  tree  yields  on  an  average  from  27  to  54  Grammes 


8  Rein  gives  2  m.m.  wide,  but  Wagner  gives  6  m.m.     Doubtless  the 
latter  is  more  nearly  correct.     v 

7  Dingler's  Polytechnisches  Jour.  218,  p.  361—1875. 


// 


» 

-'  HE 

VE^S'TY 


of  raw  lac.  In  China  the  yield  is  said  to  be  much  less,  in  some 
districts  not  more  than  10  Grammes. 

Yoshida8  states  that  Ki-Urushi  is  never  sent  to  the  market 
in  the  form  in  which  it  is  obtained  from  the  tree  but  is  usually 
mixed  with  about  40%  of  "Mokuyiki"  (wood  juice)  which  close- 
ly resembles  Ki-Urushi  but  contains  a  much  larger  proportion 
of  gum  and  about  j4  as  niuch  substance  soluble  in  alcohol.  It 
is  doubtless  an  impure  form  of  urushi  juice.  Before 
the  raw  lac  is  ready  for  use  it  must  be  strained 
through  cotton  or  linen  cloth  to  remove  pieces  of  bark  and 
foreign  particles.  It  is  then  stirred  in  a  shallow  wooden 
pail  to  remove  the  grain  and  give  it  a  uniform  consistence. 
The  varnish  makers  sometimes  add  linseed  oil ;  also  from  i — 10% 
of  perilla  oil  is  sometimes  added.  The  lac  mixed  with  1/5  perilla 
oil  is  sometimes  used  for  coating  umbrellas  and  water  proofs. 
Various  colors  are  made  by  adding  pigments.  The  red,  so  fre- 
quently used  for  a  part  of  Japanese  decorations  is  formed  by 
mixing  70  parts  Ki-Urushi,  20  parts  linseed  oil  and  10  parts  ver- 
million.  One  per  cent  of  gamboge  either  in  powder  or  in  solution 
is  sometimes  added.  The  best  gloss  black  is  formed  by  mixing 
purified  lac  with  acetate  of  iron,  formed  by  macerating  nails  or 
iron  filings  in  vinegar  or  rice  beer,  and  heating  or  exposing  to 
the  action  of  the  sun.  The  lac  thus  prepared  contains  from  0.5 
to  2%  of  iron.  Other  substances  are  sometimes  added  as  indigo, 
iron  oxide,  lead  oxide,  charcoal,  and  for  decorative  purposes,  gold 
and  silver  dust,  gold,  silver,  and  tin  foil  are  used. 

The  only  substance  used  by  the  Japanese  to  thin  the  lac  is 
camphor,  which  is  powdered  and  mixed  with  the  lac.  Rein  ob- 
served that  when  water  is  mixed  with  lac  that  it  thickens  and  be- 
comes jelly-like,  and  if  applied  to  wood  dries  very  rapidly. 

If  the  lac  is  allowed  to  harden  in  a  dry  atmosphere  it  has 
a  dull  appearance.  Hence  it  must  be  dried  in  the  presence  of 
moisture  which  is  necessary  to  ensure  the  best  action  of  the 
enzyme.  Therefore  the  articles  coated  with  the  lac  are  placed 
in  a  room  and  wet  cloths  are  hung  on  the  wall  or  about  the  lac- 
quered articles.  A  temperature  of  from  20°  to  30°  is  best  adapted 
for  this  process. 


8  Jour.  Chem.  Soc.,  1883—9.  472. 


—  10  — 


INSTRUMENTS   USED    IN   COU,ECTING   AND    APPLYING  THE   I<AC. 


INSTRUMENTS   USED  IN   COLLECTING  AND  APPLYING  THE  LAC. 

The  accompanying  reproduction  is  from  "Rein's  Japan," 
and  is  from  original  instruments  in  the  Royal  Industrial  Art 
Museum  in  Berlin. 

No.  i  is  a  sharp  kitchen  knife ;  No.  2,  a  gouge  or  chisel ; 
No.  3,  shears ;  Nos.  4  and  5,  wooden  spatulas ;  No.  6,  a  bamboo 
spatula;  No.  7,  a  surface  brush  made  from  human  hair;  No.  8, 
palette  made  of  tortoise  shell  or  buffalo  horn,  to  be  carried  on  the 
hand ;  No.  9,  spoon  for  putting  on  the  gold  or  silver  dust.  The 
above,  with  various  sized  brushes  made  of  deer  and  rat's  hair 
form  the  implements  used  in  applying  the  lac,  gold,  silver  leaf, 
etc.  Nos.  10  and  11  are  used  in  collecting  the  lac  and  have  al- 
ready been  described. 

RULES  TO  BE  OBSERVED  BY  THE  WORKMEN. 

1.  The  surface  must  be  brushed  over  equally,  first  in  one 
direction  and  then  in  the  other. 

2.  The  old  coat  must  be  thoroughly  dried  before  a  new 
one  is  applied. 

3.  The  drying  is  best  conducted  in  vapor  of  steam. 

4.  Only  the  ground  coat  can  be  dried  in  the  open  air  or 
sun,  and  that  only  when  the  coating  contains  very  little  or  no  lac. 

5.  The  peculiar  lac  properties  result  from  drying  without 
heat  in  a  chest  or  closed  room  with  wet  cloths  placed  by  the 
side  of  the  object  or  on  the  sides  of  the  room. 

6.  Dust,  light  and  air  are  to  be  excluded  while  the  varnish 
is  hardening. 

7.  For  fine  finish  the  varnish  must  be  strained  once  or  twice 
through  fine  porous  bast  or  paper. 

8.  After  every  new  coat  the  surface  should  be  polished  with 
charcoal  or  burnt  horn,  usually  with  free  application  of  water. 

9.  The  finished  object  must  not  show  the  quality  or  the 
condition  of  the  base — must  be  free  from  ridges  or  specks.    The 
complete  mirror  must  not  change  by  contact  with  hot  water. 

Wagner  states  that  the  first  coat  is  rubbed  down  with  pow- 
dered pumice  stone  and  that  the  final  coat  is  polished  with  bone 
ash  and  charcoal  and  finished  with  the  palm  of  the  hand  and  the 
tips  of  the  fingers. 


12 

CHEMICAL  INVESTIGATION  OF  LAC. 

The  most  important  chemical  investigations  of  Japanese  lac 
have  been  made  by  three  Japanese  chemists.  Ishimatsu9  made 
the  first  chemical  investigation.  He  states  that  the  lac  has  a 
sweetish  odor,  an  irritating  taste,  burns  with  a  luminous  flame 
emitting  dense  black  smoke  and  mixes  with  fixed  oils  in  all  pro- 
portions ;  hence  these  oils  are  frequently  used  as  adulterants. 

He  states  that  it  was  generally  supposed  that  the  hardening 
was  due  to  the  action  of  light  and  air,  but  he  proves  that  light 
is  practically  without  effect  on  the  lac,  as  it  is  blackened  very 
rapidly  when  exposed  to  moist  atmosphere  during  the  night  or 
when  kept  in  a  light-tight  box.  It  is  unacted  upon  even  in  sun- 
light when  kept  under  water,  or  in  carbonic  acid  in  a  sealed  flask. 
It  dries  very  slowly  in  dry  air.  This  he  attributes  to  the  rapid 
drying  of  the  surface,  which  prevents  the  evaporation  of  the 
volatile  constituents,  while  in  moist  air  the  drying  takes  place 
so  slowly  that  the  volatile  constituents  have  time  to  escape.  This 
theory  is  not  in  harmony  with  the  statement  which  immediately 
follows,  where  he  states  that  the  hardening  in  the  atmosphere 
is,  in  all  probability,  due  to  the  oxygen  of  the  air. 

He  finds  that  the  fresh  lac  yields  58.24%  of  substance  soluble 
in  alcohol,  while  the  perfectly  dry  powdered  lac  yields  only 
18.07%  °t  substance  soluble  in  alcohol.  He  attributes  this  differ- 
ence to  the  fact  that  the  alcohol  has  greater  difficulty  in  getting 
at  the  dry  lac.  He  fails  to  realize  that  the  difference  is  due  to 
a  chemical  change. 

He  reports  that  the  lac  consists  of  a  substance  soluble  in 
alcohol,  a  gum  soluble  in  hot  or  cold  water,  a  residue  insoluble 
in  alcohol  or  water,  which  consists  of  bark,  cellulose,  dust,  etc. 
There  is  also  present  a  small  quantity  of  volatile  poison  and  water. 

His  method  of  separation  was  to  extract  the  lac  with  absolute 
alcohol,  evaporate  the  alcohol  and  dry  at  100°  C.  to  constant 
weight. 

The  residue  insoluble  in  alcohol  was  extracted  with  hot 
water,  filtered  and  the  filtrate  evaporated,  dried  at  100°  C.  and 
weighed  as  gum. 


9  Chemical  investigation  of  Japanese  Laquor,  or  Urushi.  Manches- 
ter Literary  and  Philosophical  Soc.  3  series,  1882,  p.  249.  Communicated 
by  Professor  Roscoe,  Read  Feb.  18,  1879. 


—  13  — 


The  residue  insoluble  in  water  was  dried  at  100°  C.  and 
weighed. 

The  water  and  volatile  matter  were  determined  by  difference. 

Yoshida10  used  Ishii-natsu's  method  for  the  separation  of  the 
constituents,  but  reported  the  part  soluble  in  alcohol  as  urushic 
acid,  and  that  soluble  in  hot  water  as  gum,  identical  with  acacia, 
and  that  which  was  insoluble  in  water  or  alcohol  as  diastatic 
matter. 

He  proved  that  the  hardening  of  the  lac  was  due  to  the  action 
of  an  oxidizing  enzyme,  acting  in  the  presence  of  moisture.  He 
states  that  the  enzyme  is  an  albuminous  body,  coagulated  by 
boiling.  In  this  he  is  mistaken  for  the  enzyme  is  intimately  asso- 
ciated with  the  gum  and  cannot  be  separated  from  it,  even  though 
it  is  destroyed  by  boiling.  See  under  Gum,  p.  45. 

Later  Korshelt  and  Yoshida11  examined  several  samples  of 
lac,  using  the  same  method  of  separation.  For  comparison  the 
results  of  these  chemists  are  given  in  following  tabulated  form: 


CONSTITUENTS 
OF  RAW  LAC 

Yoshino 
Prov.  Yamato 
H.  Yoshida 

Korschelt  and  Yoshida 

Bought  in  Tokio 
S.'  Ishimatsu 

Hottamnaa, 
Prov.  Ilidachi 

Southern 
Sagaimi 

Northern 
Echtgo 

Hachioji, 
Prov.  Sagami 

Bought  in 
Tokio 

Lac   ?cid    (Urushic  acid)  

85.15 
3-15 
2.28 

? 

9.42 

64.62 
5.56 
2.  ID 
O.O9 
27.63 

68.83 
5-02 
2.01 
O.O6 
24.08 

66.Q2 
4-75 
1.72 
O.o6 
26.55 

80.00 
4.69 

3-31 

p 

12.00 

64.07 
6.05 
3-43 
0.23 
26.22 

58.24 
6-32 

2.27 
? 

33-17 

Oum                             

Nitrogenous  residue           

Oil    

Water        

It  is  claimed  that  the  small  quantity  of  oil  reported  is  not  a 
natural  constituent,  but  is  due  to  the  oil  of  Perilla  that  is  used 
on  the  knife  and  spatula  to  prevent  the  lac  adhering  to  the  iron. 

Yoshida  states  that  the  Yoshino  sample  which  he  analyzed 
was  collected  under  official  inspection  for  chemical  investigation 
and  was  evidently  pure  and  that  the  sample  analyzed  by  Ishimat- 


10  Chemistry    of    Lacquer,    Ki-Urushi.      Hikorokuro    Yoshida,    Jour. 
Chem.  Soc.  1883,  p.  472. 

11  Trans.  As.  Soc.  Japan,  12,  pp.  182  to  220. 


—  14  — 

su  must  have  contained  a  considerable  quantity  of  Mokuyki,  an 
impure  form  of  urushi  juice. 

Ishimatsu  states  that  the  part  soluble  in  alcohol  has  the  same 
odor  as  the  original,  but  never  dries  up  as  that  does.  It  is  brown- 
ish-black, slightly  sticky  to  the  touch.  With  potassium  hydrox- 
ide it  forms  a  bluish-black  precipitate.  The  alcoholic  solution 
was  precipitated  by  lead  acetate.  The  precipitate  was  washed, 
dried  and  analyzed,  with  the  following  results: 

MEAN 

C   49-84  51-06  50.45 

H    5.81  5.60  5.705 

O  40.-30  39-84  40.07 

PbO    3-50  4-05  3-775 

From  which  he  calculated  the  formula  C20H30O2. 

When  he  boiled  the  alcoholic  residue  with  nitric  acid  it  gave 
off  brown  fumes  and  formed  an  orange  colored  mass,  which  when 
washed  was  partly  soluble  in  absolute  alcohol.  This  alcoholic  so- 
lution formed  a  yellow  precipitate  with  lead  acetate  or  silver  ni- 
trate. The  lead  precipitate  explodes  when  heated.  It  could  not 
be  decomposed  by  sulphureted  hydrogen  without  decomposition 
of  the  acid,  therefore  he  removed  the  lead  by  sulphuric  acid,  and 
again  precipitated  with  lead  acetate,  washed,  dried  the  precipi- 
tate, and  estimated  the  lead  as  oxide,  and  the  other  constituents 
by  combustion.  The  results  were  as  follows: 

MEAN 

C   26.77  27.10  26.93 

H    4.10  4.12           4.11 

NO   18.16  18.28  18.44 

PbO    47.41  47-43  47.42 

O    3.12  3.07           3-1 

From  which  he  calculated  the  formula  C11H20(NO2)2PbOo 
and  for  the  original  substance  C^H^C^. 

The  following  is  an  abstract  of  Yoshida's  investigation  of 
the  alcohol  soluble  portion  of  the  lac,  which  he  calls  Urushic  acid. 

It  is  readily  soluble  in  benzin,  ether,  carbon  disulphide;  less 
easily  soluble  in  fusel  oil  and  petroleum  of  high  boiling  point ;  in- 
soluble in  water;  sp.  gr.  0.9851  at  23°.  Remains  unchanged  at 
1 60°.  Above  200°  it  decomposes  slowly  with  carbonization. 
From  the  alcoholic  solution  many  salts  can  be  produced,  most  of 
which  are  slightly  soluble  in  alcohol  but  insoluble  in  water. 


—  15  — 

With  silver  nitrate  it  forms  a  fine  black  precipitate  moderate- 
ly soluble  in  alcohol,  which,  on  boiling  is  reduced  with  the  for- 
mation of  a  metallic  mirror.  Platinum  chloride,  gold  chloride, 
uranium  acetate,  and  copper  nitrate  form  precipitates  varying 
in  color  from  brown  to  black. 

He  prepared  the  lead  compound  by  precipitating  with  ace- 
tate of  lead,  washing  with  alcohol  and  then  with  boiling  water, 
drying  on  a  water  bath  and  finally  over  sulphuric  acid  in  an 
exsiccator.  He  obtained  by  analysis  the  following  results: 

THEORETICAL  FOR 
FOUND  (CuH17O«)jPb. 

C 52.08  52.4 

H 5-34  5-3 

O 10.43  10.01 

Pb 32-45  32.29 

The  lead  salt  is  gray ;  on  heating  to  100°  it  gives  off  a  pecu- 
liar odor,  turns  dark,  at  iio°-ii5°  melts  to  a  brown  mass,  and 
at  about  120°  ignites  spontaneously. 

By  adding  an  insufficient  quantity  of  ferric  chloride  he 
formed  a  voluminous  black  precipitate,  which  by  analysis  gave 
the  following  formula:  (C14H17O2)3Fe+9C14H18O2. 

By  adding  a  larger  proportion  of  ferric  chloride  he  formed 
a  compound  which  on  analysis  gave  results  corresponding  to  the 
following  formula:  (C14H17O2)3Fe+3C,4H18O2.  Both  salts 
melt  to  a  black  mass  at  io5°-no°  and  ignite  spontaneously  at 
a  somewhat  higher  temperature. 

Free  alkalies  impart  a  very  dark  color  to  the  alcoholic  sol- 
ution, which  looks  purplish-black  by  transmitted  light  and  dark 
brown  by  reflected  light.  On  exposure  to  air  it  forms  a  viscid 
compound,  rapidly  becomes  black  and  dries  up. 

Soluble  salts  of  mercury,  zinc,  nickel,  cobalt,  manganese  and 
earthy  metals  do  not  give  any  distinct  reaction. 

To  a  solution  of  the  acid  in  carbon  disulphide,  bromine  was 
gradually  added  in  excess  and  the  whole  evaporated  to  dryness 
on  a  water  bath,  the  mass  extracted  with  strong  alcohol,  and  the 
extract  again  evaporated,  whereupon  it  yielded  a  dark  semi-fluid 
mass.  This  was  examined  for  bromine  by  igniting  with  pure 
lime.  0.7060  gm.  gave  1.151  gm.  AgBr= 69.37%  °f  bromine 
agreeing  very  nearly  with  a  hexabromo  derivative  of  the  acid, 
C14H12Br0O2,  which  requires  69.36%. 


—  i6  — 

Yoshida  subjected  his  urtishic  acid  to  the  continued  action 
of  hydrochloric  acid  for  three  days,  and  obtained  a  hard  brown 
mass  which  he  cut  into  pieces,  boiled  with  water,  washed  with 
alcohol,  dried  at  100°  C.  and  analyzed.  The  results  obtained  were 
practically  identical  with  those  for  urushic  acid  as  will  be  seen 
by  the  following  comparison: 

MEAN   FOUND  FOR 
FOUND  URUSHIC   ACID 

C 77-07  77.05 

H 8.77  9.01 

He  considers  this  as  a  polymerization  product,  a  molecular 
transformation  under  the  influence  of  strong  hydrochloric  acid 
and  names  it  ^-urushic  acid,  and  states  that  it  is  soluble12  in  the 
usual  solvents  for  urushic  acid.  He  claims  that  the  substance 
obtained  by  the  decomposition  of  an  alkali  salt  of  urushic  with 
hydrochloric  acid  is  the  same  body  as  ^-urushic  acid. 

By  the  action  of  strong  nitric  acid  he  obtained  a  sponge-like 
body  which  he  washed  with  water,  dissolved  in  alcohol,  and  pre- 
cipitated with  ferric  chloride.  The  precipitate  was  washed,  dried 
and  analyzed  with  the  following  results : 

CALCULATF.D   FOR 

FOUND  [C14H15(NO2)2O2]sFe. 

C 51.49  51-59 

H 4.82  4.61 

NO2 28.16  28.25 

Fe 9.77  9-8i 

This  nitro  body  was  light  yellow,  and  soluble  in  the  usual 
solvents  for  urushic  acid. 

Yoshida  oxidized  urushic  acid  with  strong  chromic  acid. 
The  product  was  washed  with  water  and  then  with  absolute  alco- 
hol and  dried  at  105°  C.  It  was  in  the  form  of  a  brown  powder 
which  by  analysis  was  found  to  contain  one  more  atom  of  oxy- 
gen than  urushic  acid.  See  results  below. 

He  heated  a  portion  of  the  fresh  juice  on  the  water  bath 
until  the  water  was  entirely  removed,  and  at  the  same  time  the 
action  of  the  enzyme  was  destroyed.  This  was  then  analyzed. 
For  results  see  below. 

Another  portion  of  the  lac  was  allowed  to  harden  in  the 
usual  way  by  the  action  of  the  enzyme  and  then  analyzed.  For 


12  Doubtless  this  is  a  typographical  error,  and  should  read  "insoluble 
in  the  usual  solvents  for  urushic  acid." 


—  i/- 
convenience  of  comparison  these  results  are  tabulated,  as  fol- 
lows: 

Mean  raw  lac     Mean  for  lac  hard-    Mean  for  urushic    Calculated  for 
dried  by  heat.      ened  by  enzyme.      acid  oxidized  by  C,4Hi8Oa 

chromic  acid. 

C    75-47  70.85  71.52  71-79 

H  8.97  8.22  8.23  7-69 

N    o.n  o. 092  ....  .... 

Ash    0.21  0.032  

0   15.17  20.52  20.25  20.52 

He  concludes  from  the  above  analyses  that  the  lac  when 
hardened  in  the  usual  manner  takes  up  one  atom  of  oxygen  for 
every  molecule  of  urushic  acid,  becoming  C14H18O3.  This  com- 
pound he  names  Oxyurushic  acid. 

More  recently  Bertrand13  has  worked  upon  Japanese  lac ; 
however  he  has  contributed  nothing  of  importance  to  the  knowl- 
edge of  the  alcohol  soluble  substance.  His  principal  work  was 
upon  the  soluble  ferment  referred  to  elsewhere. 

EXPERIMENTAL   INVESTIGATION. 

Three  samples  of  lac  were  used  in  the  following  experiments. 
The  first  and  second  were  in  glass  jars  bearing  original  Japanese 
labels.  The  third  was  in  a  tin  can.  Apparently  all  were  identi- 
cal. 

The  samples  were  all  sent  gratuitously  to  Prof.  Tschirch. 
The  first  by  forester  Shirasawa,  in  Tokio,  and  others  by  the 
Rhus  Company  in  Frankfort,  a.  M.  to  whom  I  here  extend  thanks. 

When  separated  according  to  the  method  of  Ishimatsu  they 
gave  the  following  results: 

Parts  soluble  in  alcohol 72.40% 

Parts  soluble  in  water 4.05% 

Insoluble  residue  2 . 35% 

Water  and  volatile  matter 21 .20% 

1  have  found,  as  will  appear  later,  that  Yoshida's  Urushic 
acid  may  be  separated  by  benzini    into 

Benzin-soluble    78% 

Benzin-insoluble    22% 

and  that  the  benzin  soluble  consists  of  three   substances,   one 
of  which  is  a  non-volatile  poison,  also  that  the  gum  and  enzyme 


13  Ann.  chem.  phys.  sen  XII,  1897,  p.  115. 


(diastatic  matter)  cannot  be  separated,  also  that  the  lac  contains 
acetic  acid.  The  lac  is  of  a  grayish  color.  On  exposure  to  air 
it  rapidly  darkens,  but  if  undisturbed  an  impervious  membrane 
soon  forms  on  the  surface  thus  preventing  further  change.  The 
blackening  of  the  lac  is  due  to  the  action  of  oxydase  or  "Lac- 
case,"  a  soluble  oxydizing  enzyme,  in  the  presence  of  moisture. 
The  lac  may  also  be  darkened  by  other  means,  as,  by  the  action 
of  alkalies.  For  example,  a  piece  of  wood  was  coated  with  fresh 
lac;  a  second  piece  was  coated  with  lac,  sterilized  by  suspending 
a  tube  containing  lac  in  boiling  water  for  half  an  hour;  and  a 
third  was  coated  with  sterilized  lac  containing  a  small  portion 
of  potassium  hydroxide ;  each  piece  of  wood  was  covered  with 
wet  filter  paper.  The  first  rapidly  changed  to  a  dark  brown  color 
and  in  24  hours  the  coating  was  black  and  hard.  The  second 
remained  unchanged.  The  third  immediately  changed  to  black 
but  remained  moist  for  several  days. 

LACRESINS,  THE  URUSHIC  ACID  OF  YOSHIDA,  THE;  LACCOIy  OF 
BERTRAND. 

It  was  desirous  to  separate  the  resinous  portion  from  the 
gum  and  enzyme  with  the  least  exposure  to  the  air,  therefore 
the  can  containing  lac  was  connected  with  a  flask  by  means  of 
a  tube  extending  to  the  bottom  of  each.  The  flask  was  partially 
filled  with  alcohol  and  the  lac  drawn  into  the  alcohol  by  suction. 
The  contents  of  the  flask  were  then  agitated,  filtered  and  the 
residue  exhausted  with  alcohol.  The  alcoholic  solution  was 
strongly  acid  and  had  a  peculiar  aromatic  odor  which  upon  evap- 
oration of  the  alcohol  suggested  the  odor  of  acetic  acid.  The 
oily  residue  left  after  distillation  of  the  alcohol  was  washed  by 
shaking  out  with  water.  The  watery  solution  was  neutralized 
with  potassium  hydroxide  and  heated,  when  a  fine  black  precipi- 
tate formed.  This  was  removed  by  filtration  and  the  filtrate  evap- 
orated to  dryness.  The  residue  with  sulphuric  acid  gave  an  un- 
mistakable odor  of  acetic  acid,  also  when  heated  with  sulphuric 
acid  and  alcohol  the  odor  of  acetic  ether  was  developed.  It  also 
gave  the  cacodyl  odor  when  heated  with  alkali  and  with  arsenic 
trioxide.  To  prove  that  the  presence  of  acetic  acid  was  not  due 
to  the  oxidation  of  the  alcohol,  a  fresh  portion  of  lac  was  ex- 
tracted with  ether,  and  the  ether  residue  treated  as  above  with 
the  same  results.  It  is  therefore  evident  that  the  lac  contains  acetic 


—  19  — 

acid.  The  black  precipitate  which  separated  from  the  watery  solu- 
tion on  evaporation  was  washed,  and  upon  adding  hydrochloric 
acid,  changed  to  a  red  color,  but  remained  insoluble  in  all  ordinary 
solvents.  There  was  not  a  sufficient  quantity  for  analysis,  but 
doubtless  was  the  same  substance  which  is  formed  as  often  as 
the  lac  is  oxidized  and  henceforth  will  be  designated  as  oxyurush- 
in. Its  presence  in  the  water  solution  was  due  to  the  acetic  acid, 
which  is  an  excellent  solvent  for  the  unoxidized  lac. 

One  hundred  grammes  of  the  lac  was  placed  in  a  flask  and 
steam  passed  through  the  lac  for  several  hours.  The  distillate 
was  covered  with  a  thin  oily  film,  which  was  removed  by  shak- 
ing out  with  ether  and  evaporating.  The  residue  was  not  volatile 
or  poisonous.  Besides  this  the  distillate  contained  acetic  acid. 

A  portion  of  the  alcoholic  residue  was  dissolved  in  ether 
and  repeatedly  shaken  out  with  one  per  cent  solution  of  sodium 
carbonate.  The  carbonate  solution  was  at  first  green, 
then  brown.  This  was  heated  on  a  steam  bath  to  remove  the  dis- 
solved ether,  and  acidulated  with  hydrochloric  acid,  when  a  small 
quantity  of  reddish  brown  precipitate  appeared.  This  was  wash- 
ed and  dried.  Only  a  small  part  of  it  was  soluble  in  ether,  the 
remainder  being  insoluble  in  any  of  the  ordinary  solvents,  and  is 
evidently  oxyurushin.  ,. .,.,. 

The  ether  solution  which  was  separated  from  the  carbonate 
solution  was  next  shaken  out  with  one  per  cent  potassium  hy- 
droxide solution.  The  resulting  solution  was  very  dark  green, 
changing  later  to  brown.  It  was  treated  exactly  as  in  the  case 
of  the  carbonate  solution  and  with  the  same  result,  i.  e.  a  sepa- 
ration of  the  same  insoluble  oxyurushin. 

The  ether  solution  then  received  attention.  It  was  shaken 
out  with  5%  potassium  hydroxide,  producing  a  black  precipitate 
which  floated  in  the  ether  solution.  The  alkaline  solution  was 
again  of  the  same  dark  green  color  changing  to  brown,  and 
when  acidulated  produced  the  same  insoluble  oxyurushin.  The 
black  precipitate  was  removed  from  the  ether  by  filtration  and 
the  ether  shaken  with  more  alkali  with  the  same  result.  By  re- 
peating the  operation  a  sufficient  number  of  times,  the  entire 
substance  could  be  removed  from  the  ether.  The  black  precipi- 
tate formed  by  potassium  hydroxide  was  washed  free  from  alkali 
and  heated  with  dilute  hydrochloric  acid,  washed,  dried  and  pow- 
dered ;  a  very  small  part  of  it  was  soluble  in  ether  but  this  became 
insoluble  on  evaporation.  The  powder  was  reddish  brown  and 


2O  — 


in  every  way  identical  with  oxyurushin.  The  alkaline  solutions 
from  which  the  oxyurushin  was  precipitated  by  acid,  were  evap- 
orated and  extracted  with  ether  and  alcohol  but  no  organic  sub- 
stance was  obtained. 


SEPARATION  BY  LEAD  ACETATE  AND  SUBACETATE. 

Another  part  of  the  alcoholic  residue,  free  from  acetic  acid, 
was  redissolved  in  alcohol  and  an  alcoholic  solution  of  lead  ace- 
tate added  as  long  as  it  formed  a  precipitate.  The 
precipitate  which  was  of  a  light  gray  color  was  washed  with 
alcohol,  mixed  with  fresh  alcohol,  decomposed  with  sulphuric 
acid,  and  the  excess  of  acid  removed  by  shaking  with  lead  car- 
bonate. On  evaporating  the  alcohol  a  thick  dark  brown  oily  resi- 
due was  obtained.  The  residue  was  somewhat  darker  than  the 
original  alcoholic  residue,  but  otherwise  similar.  To  the  nitrate 
secured  from  the  lead  acetate  precipitate,  lead  subacetate  was 
added  as  long  as  a  precipitate  formed.  The  precipitate  was  of 
a  gray  color,  but  a  decidedly  lighter  gray  than  that  obtained  by 
lead  acetate.  On  decomposing  the  precipitate,  as  above,  an  oily 
residue  was  obtained,  which  was  also  lighter  in  color  than  that 
obtained  from  the  lead  acetate  residue. 

The  filtrate  from  the  subacetate  precipitate  was  still  of  a 
brownish  color.  The  excess  of  lead  was  removed  by  adding  a 
slight  excess  of  sulphuric  acid,  and  the  excess  of  acid  removed 
by  shaking  with  lead  carbonate  and  filtering.  The  filtrate  was 
concentrated  by  evaporation  and  shaken  out  with  ether.  Upon 
evaporating  the  ether  a  residue  was  obtained  which,  when  dis- 
solved in  alcohol,  was  readily  precipitated  by  lead  acetate  or  sub- 
acetate.  By  repeated  experiments  with  the  original  alcoholic 
solution  it  was  found  that  by  precipitation  with  lead  acetate  and 
removing  the  lead  and  acid  from  the  filtrate,  reprecipitating  and 
continually  repeating  this  operation,  that  a  series  of  oily  residues 
could  be  obtained,  gradually  diminishing  in  quantity,  and  each 
increasing  in  fluidity,  and  becoming  a  shade  lighter  than  the  pre- 
ceding. Only  the  last  fractions  were  poisonous. 

Lead  subacetate  is  a  better  precipitant  than  the  acetate.  The 
acetic  acid  liberated  evidently  aids  in  preventing  complete  pre- 
cipitation. The  fact  that  the  fractions  decrease  in  color  and 
viscosity,  and  that  only  the  last  were  poisonous,  indicates  that  the 
alcoholic  extract  consists  of  a  mixture  of  two  or  more  substances. 


—  21  — 

But  in  no  case  can  the  above  method  be  considered  as  a  com- 
plete separation.  In  alcoholic  solutions  each  fraction  assumed 
a  green  or  greenish  black  color  with  alkalies,  the  color  varying 
with  the  concentration  of  the  solution  and  the  strength  of  the 
alkali  used. 

SEPARATION  OF  THE  LACRESINS  BY  SOLVENTS. 
SOLUBILITY  OF  THE  ORIGINAL  ALCOHOLIC  RESIDUE. 

On  first  trial  it  appeared  as  though  the  alcoholic  residue 
might  be  soluble  in  any  of  the  ordinary  solvents  for  oils  and 
resins,  but  investigation  proved  that  it  was  not  completely  soluble 
in  carbon  disulphide,  methyl  alcohol,  amyl  alcohol  or  petroleum 
benzin  in  all  proportions. 

After  numerous  experiments  the  following  method  of  pro- 
cedure was  adopted.  The  alcoholic  residue  was  dissolved  in  the 
proportion  of  i  part  to  7  of  petroleum  benzin,  boiling  point  not 
over  60° C.,  forming  a  clear  solution,  but  further  addition  of  ben- 
zin caused  a  precipitate.  This  was  then  poured  into  55  parts  of 
benzin  which  produced  the  immediate  separation  of  a  thick  brown 
mass  from  which  the  still  cloudy  benzin  was  decanted. 
The  brown  deposit  was  dissolved  in  a  small  quantity  of  benzin  and 
again  separated  by  adding  a  larger  amount.  After  this  operation 
was  repeated  several  times  the  deposit  became  entirely  insoluble 
in  benzin.  It  was  then  washed  with  benzin  until  the  last  washings 
were  colorless.  The  washings  were  added*  to  the  portions  pre- 
viously decanted  and  allowed  to  stand  12  hours — when  the  ben- 
zin became  clear  and  was  not  affected  by  the  further  addition  of 
benzin.  The  second  deposit  was  dissolved,  again  precipitated  by 
benzin  and  washed  by  agitation  with  benzin.  The  second  deposit 
was  thinner  and  lighter  in  color  than  the  first. 

By  this  method  the  alcoholic  residue  was  separated  into  two 
distinct  substances,  a  benzin-soluble,  and  a  benzin-insoluble. 
The  first  and  second  deposit  from  benzin,  while  differing  some- 
what in  physical  appearance  could  not  be  said  to  consist  of  two 
distinct  substances  but  doubtless  consisted  of  mixtures  of  the 
same  substances  in  varying  proportions.  Each  was  mixed  with 
a  small  quantity  of  ether  and  added  to  methyl  alcohol  which  im- 
mediately became  cloudy  and  on  standing  a  short  time  formed  a 
deposit.  The  solution  remained  clear  on  the  further  addition  of 
methyl  alcohol.  The  residue  was  separated  and  washed  with 


—  22 — 

methyl  alcohol  but,  as  the  washings  remained  turbid  for  several 
days,  they  were  not  added  to  the  original  solution. 

The  first  benzin  deposit  contained  more  substance  insoluble 
in  methyl  alcohol  than  the  second  deposit,  otherwise  no  differ- 
ence appeared,  therefore  their  products  were  combined  as  methyl 
alcohol-soluble  and  methyl  alcohol-insoluble  substances.  About 
half  of  the  methyl  alcohol-insoluble  was  soluble  in  ether,  the 
remainder  apparently  having  undergone  some  change  during 
manipulation  with  the  methyl  alcohol.  This  theory  is  supported 
by  the  fact  that  the  methyl  alcoholic  solution  slowly  deposits  on 
standing.  A  similar  condition  was  also  observed  when  a  por- 
tion of  the  original  alcoholic  residue  was  precipitated  with  pe- 
troleum benzin  as  a  small  portion  of  this  also  remained  insoluble 
in  ether.  In  fact  all  of  the  substances  so  far  separated  are  evi- 
dently slowly  oxidized,  as  all  solutions  except  the  petroleum 
benzin  solution  on  standing  for  weeks  form  a  slight  insoluble 
deposit ;  and  while  the  soluble  benzin  portion  apparently  remained 
unchanged,  yet  on  largely  diluting  with  benzin  it  again  became 
cloudy ;  whereas  previously  it  remained  clear  under  similar  condi- 
tions. 

Three  samples  of  the  benzin  soluble  substance  were  placed 
in  small  colorless  glass  vials.  No.  I  was  corked  and  wrapped 
in  black  paper,  No.  2  was  merely  corked  and  No.  3  was  loosely 
closed  with  cotton.  All  were  placed  on  a  shelf  exposed  to  strong 
light  and  allowed  to  remain  for  10  months.  When  they  were 
tested  as  to  their  solubilities  Nos.  i  and  2  dissolved  readily  in  ben- 
zin but,  on  diluting  with  a  large  amount  of  benzin,  became  cloudy 
and  upon  standing,  formed  a  slight  deposit.  That  from  No.  I, 
being  a  little  larger  than  from  No.  2,  would  indicate  that  the 
reducing  action  of  light  has  a  tendency  to  prevent  the  change. 
No.  3  became  quite  thick  but  was  still  fluid.  Only  a  small  part 
was  soluble  in  benzin. 

SOLUBILITY  OF  THE  SUBSTANCES  SEPARATED  FROM  THE  ALCOHOLIC 

RESIDUE. 

The  part  soluble  in  benzin  was  also  soluble  in  ether,  chloro- 
form, alcohol,  methyl  alcohol,  amyl  alcohol,  carbon  disulphide, 
toluol,  oxylol,  acetone,  toluidin,  pyridin,  quinolin,  carbon  tetra- 
chloride,  amyl  acetate,  acetic  ether,  nitro  benzol,  turpentine  oil, 
acetic  acid  and  80%  solution  of  chloral  hydrate. 


The  part  soluble  in  methyl  alcohol  was  soluble  in  each  of  the 
above  except  benzin.  The  part  insoluble  in  methyl  alcohol  but  sol- 
uble in  ether,  was  insoluble  in  benzol,  toluol,  oxylol,  alcohol,  amyl 
alcohol,  carbon  disulphide,  turpentine,  carbon  tetrachloride,  ace- 
tic acid  and  chloral  hydrate  solution. 

The  part  insoluble  in  ether  was  also  insoluble  in  any  of  the 
above  solvents. 

SEPARATION    OF   THE   BENZIN-SOUJBLE   PORTION. 

One  volume  of  the  substance  was  dissolved  in  eight  volumes 
of  benzin,  four  volumes  of  alcohol  added  and  the  whole  thor- 
oughly agitated.  Upon  standing  two  layers  appeared.  The  upper 
benzin  layer  was  of  a  yellowish  brown  color,  the  lower  reddish 
brown.  These  were  separated  and  the  benzin  solution  washed 
with  alcohol  as  long  as  any  thing  could  be  removed.  The  benzin 
was  driven  off  by  evaporation  leaving  a  non-poisonous,  oily, 
brown  residue,  insoluble  in  alcohol. 

The  alcoholic  solution  was  in  turn  washed  by  shaking  out 
with  benzin  but  with  no  positive  result.  The  solution  was  next 
evaporated  and  a  reddish  brown,  slightly  gelatinous  residue  ob- 
tained. By  rapidly  washing  this  with  a  small  quantity  of  benzin 
and  evaporating  a  clear,  light  reddish  brown  residue  was  secured. 
Both  of  these  residues  proved  to  be  poisonous.  By  continual 
washing  with  benzin  or  by  employing  it  in  larger  quantities,  the 
entire  residue  was  dissolved.  I  believe  that  this  residue  consists 
of  a  poisonous  and  a  non-poisonous  substance  but  thus  far  the 
separation  has  not  been  secured.  I  hope  to  be  able  to  separate 
them  later. 

All  of  the  resins  separated  from  the  lac  were  tested  for 
cholestrin  but  with  negative  results. 

CHEMICAL   REACTIONS. 

All  of  the  substances  separated  gave  precipitates  with  lead 
acetate,  subacetate,  silver  nitrate,  mercurous  nitrate,  cupric  ace- 
tate and  ferric  chloride.  The  lead  precipitates  were  of  a  light 
gray  color  gradually  becoming  darker  on  standing.  The  other 
precipitates  were  black.  All  were  slowly  blackened  by  the  action 
of  concentrated  sulphuric  acid.  In  the  cold,  concentrated  nitric 
acid  colored  the  benzin-soluble  substance  red  which  changed  to 
brown  on  heating.  The  methyl  alcohol-soluble  substance  was 


slightly  darkened  by  cold  nitric  acid  but  became  lighter  on  heat- 
ing, forming  a  light  spongy  mass. 

The  benzin  and  methyl  alcohol  soluble  substances  were 
slightly  darkened  by  strong  hot  hydrochloric  acid,  but  by  continual 
heating  formed  a  spongy  mass  of  a  lighter  color.  All  substances 
except  the  ether-insoluble  were  at  first  colored  green,  then  black 
by  strong  alkalies.  Various  shades  of  green,  and  black  were  ob- 
tained, the  shades  varying  with  the  concentration  of  the  alkali 
and  of  the  substance  in  alcohol  or  ether.  Barium  and  calcium 
hydroxides  produced  the  same  effect  though  in  a  minor  degree. 

When  heated  with  dry  potassium  hydroxide  the  substances 
furnished  vapors  that  changed  red  litmus  to  blue,  but  gave  no 
odor  of  ammonia.  However  the  pyrrol  reaction  was  obtained 
when  a  pine  shaving  was  moistened  with  hydrochloric  acid  and 
held  in  the  vapors.  All  of  the  above  were  tested  for  nitrogen 
by  the  Lassaigne  test  but  with  negative  results". 

The  pyrrol  reaction  was  also  obtained  by  boiling  the  sub- 
stance with  a  strong  solution  of  potassium  hydroxide  but  the 
reaction  was  not  as  vigorous,  and  long-continued  heat  would  be 
necessary  to  convert  all  the  nitrogen  into  pyrrol.  This  is  proven 
by  the  fact  that  the  black  precipitate  still  gave  the  pyrrol  reaction, 
though  formed  by  heating  these  substances  with  $%  potassium 
hydroxide  for  two  hours  on  the  steam  bath. 

ANALYSIS   OF   SEPARATED   CONSTITUENTS. 

The  methyl  alcohol  soluble  substance  was  spread  on  glass 
and  placed  in  the  drying  oven.  After  several  days  it  was  suffi- 
ciently dry  to  be  removed  from  the  glass  with  a  knife.  The 
shavings  were  not  brittle,  but  were  cut  into  small  pieces  and 
returned  to  the  oven  where  they  remained  for  two  weeks  before 
they  could  be  pulverized.  The  particles  were  hard  and  electric 
but  by  constant  moistening  with  a  mixture  of  alcohol  and  ether 
they  were  finally  powdered.  The  powder  was  placed  in  a  nar- 
row tube  and  percolated  with  ether  which  dissolved  a  small 
amount.  The  ether  was  evaporated  and  the  residue  again  spread 
on  glass  when  in  a  few  hours  it  became  insoluble.  This  was 
doubtless  part  of  the  substance  which  had  been  prevented  from 
drying  by  the  surrounding  particles.  Some  of  the  powder  was 
ignited,  after  which  a  little  ash  was  left.  The  remainder  was 


"  See  tests  for  Nitrogen  under  gums. 


—  25  — 

repeatedly  boiled  with  hydrochloric  acid  and  washed  with  hot 
water  but  it  was  impossible  to  entirely  remove  the  ash.  When 
examined  this  was  found  to  consist  of  silica,  aluminum  and 
traces  of  calcium.  The  powder  was  dried  at  a  temperature  of 
105°  and  analyzed,  with  the  following  result: 

I    0.283    Gm.  gave  0.2045  H2O,  0.7452  Gm.  CO2 
II    0.3268  Gm.  gave  0.2288  H2O,  0.8457  Gra.  COjj 

I    0.351    Gm.  gave  5   Cc.  N  at  20°   C.  and  716  Mm. 
II    0.4619  Gm.  gave  6«9Cc.  N  at  18.6°  C.  and7i6  Mm. 

I  II  Mean 

C  71  808  per  cent  71.51    per  cent  71.659 

H  8.01    per  cent  7.85    per  cent  7.93 

N  1.57    percent  1.646  per  cent  1.608 

Ash       0.600 

Another  sample  of  the  same  substance  was  dissolved  in 
alcohol,  a  solution  of  sodium  hydroxide  added  in  excess,  and 
heated  until  the  alcohol  evaporated.  The  bulky  black  precipitate 
was  washed  until  free  from  alkali,  then  boiled  with  hydrochloric 
acid  which  changed  the  color  to  a  reddish  brown.  The  precipitate 
was  washed  until  free  from  acid,  dried,  powdered  and  analyzed 
with  the  following  results: 

I  0.2436  Gm.  gave  0.1718  Gm.  H2O,  0.6411  Gm.  COg 
II  0.222  Gm.  gave  0.1534  Gm.  H2O,  0.5814  Gm.  CO8 

I  0.2876  Gm.  gave  1.5  Cc.  N  at  2O°C.  and  711.5  Mm. 
II  0.343  Gm.  gave  2.0  Cc.  N  at  24. 5°  C.  and  714  Mm. 
I  II  Mean 

c        71.72  71.418  71-569 

H  7.788  7.773  7.830 

N  0.49  0.560  0.525 

Ash          i.  08  i.  060  1.070 

The  substance  insoluble  in  methyl  alcohol  and  insoluble  in 
ether  was  heated  with  hydrochloric  acid  for  four  hours  to  remove 
ash,  but  even  that  did  not  secure  its  complete  removal.  It  was 
washed  until  free  from  acid,  dried  and  analyzed  with  the  accom- 
panying results: 

0.388  Gm.  gave  0.2881  Gm.  H2O,  1.0232  Gm.  CO8 

I    0.554  Gm.  gave  8.4  Cc.  Nat  23°C.  and  714  Mm.=i.6  per  ct  |  Mean  1,68 
II    0.457 Gm.  gave 7. 6  Cc.  N  at  25°C,  and  712  Mm. =1.76  perct  f    percent 
C        71.896 
H         8.303 
N          i.  680 
Ash      0.400 

The  substance  insoluble  in  methyl  alcohol  but  soluble  in  ether 
was  spread  on  glass,  placed  in  the  drying  oven  where  it  dried  in  a 


—  26  — 

few  hours.    It  was  very  hard  to  powder  and  was  insoluble  in  all 
ordinary  solvents.    When  analyzed  it  gave  the  following  results: 

0.3606  Gm.  gave  0.295  Gm.  H2O,  0.971  Gm.  CO2 
0.4104  Gin.  gave  7.3  Cc.  at  25°  C.  N  and  712  Mm. 

C          73430 
9-J45 

N  1.850 

Ash       0.451 

A  second  sample  of  the  above  was  dissolved  in  ether  and 
alcohol  added,  then  precipitated  with  sodium  hydrate,  heated 
for  two  hours  on  the  steam  bath,  washed  until  free  from  alkali, 
again  heated  for  four  hours  writh  hydrochloric  acid,  washed  and 
dried.  Analysis  gave  the  following  result: 

0.2074  Gm.  gave  0.194  Gm.  H2O,  0.5482  Gin.  CO2 
0.2926  Gm.  gave  2.2  Cc.  N  at  21°  C.  and  712.5  Mm. 
C         72.08 
H         10.46 
N  0.74 

Ash       1.02 

Part  of  the  benzin-soluble  substance  was  dissolved  in  alcohol, 
a  strong  solution  of  sodium  hydroxide  added  and  heat  applied. 
An  adhesive,  black  mass  formed  on  the  surface  with  a  thick  gray- 
ish-black mixture  underneath.  When  cold,  globules  of  an  oily  sub- 
stance could  be  seen.  An  attempt  was  made  to  separate  them  but 
without  success.  Ether  and  benzin  dissolved  a  large  part  of  the 
precipitate  together  with  the  oily  globules,  forming  an  insepar- 
able emulsion.  More  sodium  hydroxide  was  added  and  the  whole 
again  heated  for  two  hours,  when  a  black  precipitate  separated 
leaving  a  clear  solution.  The  solution  was  separated  as  com- 
pletely as  possible  by  decantation.  Distilled  water  was  added 
to  the  precipitate,  when  it  again  formed  a  homogeneous  mixture 
as  inseparable  as  before — but  the  addition  of  more  alkali  pro- 
duced a  separation.  All  attempts  to  free  the  precipitate  from 
alkali  by  washing  failed,  as  it  invariably  formed  the  same  homo- 
geneous mixture  which  could  not  be  filtered  and  refused  to  sep- 
arate on  standing.  However,  the  addition  of  sodium  chloride 
to  the  mixture  secured  a  perfect  separation.  The  precipitate  was 
carefully  collected  on  a  filter  and  washed  with  solution  of  sodium 
chloride  until  nearly  free  from  alkali.  It  was  then  dried  and 
heated  with  hydrochloric  acid  which  changed  the  color  to  reddish 
brown.  The  precipitate  was  washed  until  free  from  acid,  dried 
and  powdered.  The  powder  was  exhausted  with  ether  which 


—  27  — 

dissolved  about  one-third.  The  insoluble  part  which  continued 
obstinately  insoluble  in  all  ordinary  solvents,  was  dried  and  anal- 
yzed. Result  of  analysis : 

I  0.366    Gra.  gave  0.2628  Gm.  H2O,  0.945    Gm.  CO2 

II  0.3016  Gm.  gave  0.213    Gm.  H2O,  0.7855  Gm,  CO« 

I  0.3566  Gm.  gave  I  Cc.  N  at  21°  C.  and  717  Mm. 
II  0.3006  Gm.  gave  I  Cc.  N  at  22°  C.  and  719.5  Mm. 

I  II  Mean 

C         70.96  7I-032  70.996 

H           8.03  7.899  7.965 

N           0.24  0.25  0.245 

Ash        1.200 

The  ether  solution  was  darker  and  more  truly  red  than  any 
of  the  other  solutions ;  on  allowing  the  ether  to  evaporate  spon- 
taneously and  allowing  the  residue  to  stand  for  a  few  hours,  a 
portion  became  insoluble  in  all  ordinary  solvents.  This  was 
exhausted  with  ether,  dried  and  analyzed  with  the  following 
results : 

I    0.3104  Gm.  gave  0.2387  Gm.  H2O,  0.7983  Gm.  CO8 

II  0.2394  Gm.  gave  0.19      Gm.  H2O,  0.622    Gm.  COjj 

I    0.415    Gm.  gave  3.2  Cc.  N  at  21°  and  720  Mm. 
I  II  Mean 

C        71.012  70.897  70-954 

H         8.596  8.877  8.736 

N 0.850 

Ash 0.210 

The  ether  extract  from  above  was  allowed  to  evaporate 
spontaneously  as  before  but  remained  soluble  in  ether  and  alcohol 
even  after  standing  several  days  and  after  repeated  solution  and 
evaporation.  On  heating  the  residue  at  100°  for  a  short  time  it 
became  insoluble  and  behaved  in  all  respects  like  the  preceding. 
Analysis  gave  these  results: 

I    0.329    Gm.  gave  0.2501  Gm.  H2O,  0.8874  Gm.  CO2 
II    o  3374  Gm.  gave  0.2588  Gm.  H8O,  0.9121  Gm.  CO2 

I    0.3946  Gm.  gave  3.4  Cc.  N  at  22°  C.  and  720  Mm. 
I  II  Mean 

C  73-554  73-721  73.637 

H  8.503  8.579  8.541 

N  1x940  0.940 

Ash                                          o.i  60 

H.  Yoshida  oxidized  the  alcoholic  extract  (Urushic  acid) 
with  chromic  acid  mixture,  and  obtained  a  brownish  powder 
which  he  washed  with  alcohol,  dried  and  analyzed. 


—  28  — 


For  comparison  his  results  will  be  given  with  the  means 
from  the  preceding  results: 

PART  INSOLUBLE  IN  BENZIN  BUT  SOLUBLE  IN  METHYL  ALCOHOL. 


C 

H 

N 

Ash 

I     Methyl  alcohol  soluble  (dried) 

7i  659 

7  93O 

I  608 

o  600 

2     Methyl  alcohol  soluble  precip.  by  NaOH  

7L569 

7.830 

0.525 

1.070 

PART  INSOLUBLE  IN  METHYL  ALCOHOL. 


3 
4 
5 

6 

7 

8 

Ether-insoluble  

71.896 

73-430 
72.080 

ZIN. 

70.996 
70.954 
73.637 

8.306 

9-145 
10.460 

7.965 
8.736 
8.541 

i.  680 
1.850 
0.740 

0.245 
0.85 
0.94 

0.400 
o.45i 

1.02 

1.200 
0.210 

0.160 

Ether-soluble  (dried)  

Ether-soluble  precip.  by  NaOH  

PART  SOLUBLE  IN  BEN 
Benzin-soluble  precip.   by  NaOH  and  insol- 
uble in  ether 

Benzin-soluble  precip.  by  NaOH,  soluble  in 
ether  but  insol  on  evaporation  

Benzin-soluble  precip    by    NaOH.    soluble 
in  ether  became  insoluble  with  heat  

Yoshida's  oxy-urushic  acid 71.52    8.280 

The  methyl  alcohol  and  ether  soluble  substances  (Nos.  4  and 
5)  constitute  less  than  one  per  cent  of  the  original  alcoholic  resi- 
due, hence  not  enough  material  remained  after  other  experiments 
for  dulpicate  analyses.  Likewise  only  a  small  per  cent  of  the 
alcoholic  residue  is  represented  by  No.  8,  that  part  of  the  benzin- 
soluble  which,  after  treating  with  sodium  hydroxide  and  hydro- 
chloric acid,  required  heat  to  convert  it  into  the  insoluble  form. 
These  would  have  no  practical  effect  upon  the  final  product 
obtained  by  drying  or  oxidizing  the  lac.  The  substances  from 
which  the  results  in  numbers  I,  2,  6  and  7  were  obtained  form 
the  principal  part  of  the  lac-resin  that  is  soluble  in  alcohol. 

Yoshida  analyzed  the  dried  lac  and  found  C  70.85%, 
H  8.22%,  N. -0.092,  Ash  0.032. 

The  one  quality  which  has  made  Japanese  lac  so  valuable  is 
its  power  to  resist  atmospheric  action,  solvents  and  chemicals. 
The  preceding  results  show  that  strong  chemicals,  like  alkalies 
and  acids,  convert  the  lac-resin  into  its  insoluble  form.  Also  that 
the  final  product  appears  to  be  the  same  whether  obtained  by  the 
action  of  the  enzyme,  as  in  the  usual  method  of  hardening,  or  by 
chemical  action.  This  becomes  more  apparent  by  comparison  of 
the  composition  of  this  substance  as  obtained  by  various  methods. 

C         H         N       Ash 
Mean  from  Nos.  I  and  3  obtained  by  drying    71.777    8.1 18     1.644    0.5 

Mean  of  Nos.  2,  6  and  7  precip.  by  NaOH 71.173    8.177    0.56      0.82 

Action  of  chromic  acid  (Yoshida) 71.52      8.280     

Original  lac  hardened  (Yoshida) 70.85      8.22      0.092    0.032 


_-29  — 

Yoshida  has  named  this  substance  oxy-urushic  acid  and  giv- 
en it  the  formula  C14H18O3,  but  as  it  has  none  of  the  properties 
of  an  acid  I  have  called  it  oxy-urushin.  Owing  to  the  presence 
of  nitrogen  I  am  loth  to  suggest  a  change  in  the  formula.  That 
nitrogen  is  present  has  been  proved  (beyond  doubt)  by  the  meth- 
ods more  fully  given  under  gum-enzyme.  That  nitrogen  is  in 
actual  combination  is  supported  by  the  fact  that  it  is  separable 
only  by  fusing  with  dry  fixed  alkali,  or  incompletely  by  long 
boiling  with  a  solution  of  fixed  alkali.  Owing  to  the  small  amount 
of  nitrogen  present,  a  very  slight  error  in  estimation  would  ma- 
terially affect  the  results  from  calculation  of  a  molecular  formula. 

The  Kjeldahl  method  could  not  be  used  for  the  determina- 
tion of  the  nitrogen,  hence  the  necessity  of  using  the  Dumas 
method.  The  principal  objection  to  this  method  is  the  difficulty 
of  completely  removing  the  air  from  the  fine  copper  oxide.  After 
numerous  experiments  the  substance  was  finally  mixed  with  cop- 
per oxide  in  fine  powder  and  placed  in  a  copper  boat  which  was 
then  placed  in  the  combustion  tube  and  the  air  removed  by  car- 
bon dioxide,  generated  from  sulphuric  acid  and  potassium  car- 
bonate by  Thiele's  method15.  The  results  were  very  concordant. 

For  calculation  of  the  empirical  formula  only  those  results 
which  were  obtained  without  heating  with  alkali,  can  be  used, 
as  alkali  causes  a  loss  of  nitrogen.  Therefore  the  mean  from  I 
and  3,  corrected  for  ash,  was  used  as  follows: 

Mean  from  Calculated  for  Ash  Calculated  for 

i  and  3:  Free  Substance  CiogHiag^O^ 

C....  71.777  72.137  72.206 

H....  8.118  8.156  8.202 

N....  1.644  1.652  1.656 

Ash..  0.5  

o .*....  17.936 

The  above  formula  was  calculated  from  the  benzin-insoluble 
portion  which  represents  only  22%  of  that  portion  of  the  lac 
that  is  soluble  in  alcohol.  The  remaining  78%  which  is  soluble 
in  benzin  could  not  be  obtained  in  a  dry  form,  or  changed  into 
its  end  product  without  the  use  of  reagents  which  caused  a  loss 
of  nitrogen;  hence  no  attempt  was  made  to  calculate  a  formula 
from  the  results  obtained  by  combustion.  The  results  obtained 
in  6,  7  and  8  differ  so  much  from  those  of  2  and  3  that  it  would 


15  Ann.  der  chem.  253,  1889,  p.  242. 


indicate  a  difference  in  composition,  though  the  physical  proper- 
ties are  the  same. 

It  is  evident  that  the  urushic  acid  of  Yoshida  consists  of  at 
least  four,  if  not  five  substances,  differing  in  their  solubilities  as 
well  as  in  other  respects,  for  I  have  already  shown  that  some  dry 
in  air  to  an  insoluble  substance,  while  others  remain  for  months 
without  drying;  one  is  piosonous  and  the  others  not.  However 
it  is  a  remarkable  fact  that  all  may  be  converted  into  an  end  pro- 
duct having  practically  the  same  properties,  resisting  the  action 
of  all  ordinary  solvents,  are  black  when  heated  with  alkalies  and 
red  when  heated  with  acids.  The  product  seems  to  be  the  same 
whether  obtained  by  drying  with  heat,  by  the  action  of  the  enzyme, 
or  by  the  action  of  alkalies. 

ORIGINAL  RESIDUE  INSOLUBLE  IN  ALCOHOL  OR  WATER. 

The  residue  from  the  lac  which  was  insoluble  in  alcohol  or 
water  consisted  largely  of  hardened  lac.  By  boiling  this  with 
caustic  alkali  a  dark  brown  solution  was  obtained  which,  on  neu- 
tralization and  evaporation,  left  a  hygroscopic  residue,  soluble  in 
water  but  insoluble  in  alcohol  or  ether.  All  attempts  to  obtain 
a  crystalline  product  failed. 

The  residue  insoluble  in  alkali  was  dissolved  by  continued 
heating  with  fuming  nitric  acid,  concentrated,  and  precipitated 
by  pouring  into  water.  The  precipitate  formed  a  gummy,  plastic 
mass,  soluble  in  alcohol  but  non-crystallizable. 

The  watery  solution  contained  oxalic  acid,  but  no  picric  or 
styphnic  acid. 

Oxyurushin  when  treated  with  fuming  nitric  acid  gave  the 
same  results  as  above. 

PREVIOUS   INVESTIGATIONS  OF   SOLUBLE   FERMENTS. 

The  first  observation  recorded  upon  the  color  action  of  Gums 
and  of  guaiac  was  made  in  1809  by  Goettling18  who  observed  a 
bluish-gray  color  when  compounding  a  mixture  of  resin  of  guaiac, 
sugar,  acacia,  and  peppermint  water.  In  the  same  year  Boulay17 
observed  the  same  color  reaction  when  syrup,  gum  arabic  and  tr. 
of  guaiac  were  mixed  and  by  experiments  proved  that  the  color 


16  Bulletin  de  Pharmacie  t.  I.  p.  220,  1809. 

17  Bulletin  de  Pharmacie  t.  I.  p.  225,  1809. 


was  produced  by  the  acacia  and  guaiac.  He  also  records  the  fact 
that  certain  toothache  remedies  containing  resin  of  guaiac  colored 
the  mouth  blue  or  green,  and,  from  experiments  with  albuminous 
substances,  concluded  that  the  color  was  produced  by  the  albumen 
in  the  saliva  and  the  guaiac. 

Planche"  in  1810  observed  that  pieces  of  certain  fresh  roots 
colored  tr.  of  guaiac  blue,  also  that  nearly  the  same  effect  was 
produced  by  sulphurous  acid  gas  and  tr.  guaiac. 

In  1819  Taddey19  observed  that  corn  meal  and  powdered 
resin  of  guaiac  when  mixed  with  water  and  exposed  to  the  air 
became  blue. 

Rudolphi19  found  that  in  the  above  mixture  the  color  was 
produced  by  gluten  and  guaiac. 

Planche20  in  1820  gives  a  list  of  about  25  plants,  the  fresh 
roots  of  which  give  a  blue  color  with  tincture  of  guaiac,  and  states 
that  fresh  milk  not  boiled  produces  the  blue  color  with  tr.  guaiac. 
He  was  the  first  to  discover  that  the  power  of  albuminous  sub- 
stance to  produce  the  blue  color  with  tr.  of  guaiac  was  destroyed 
by  heat.  He  experimented  upon  the  action  of  light  and  air,  suc- 
cessively examined  and  rejected  the  intervention  of  oxygen,  but 
concluded  that  action  was  due  to  a  kind  of  undetermined  cyano- 
gen. 

Schonbein21  in  1856  observed  that  the  juice  of  certain  mush- 
rooms, Boletus  luridus  and  Agaricus  sangnincns,  colored  tincture 
of  guaiac  blue  but  lost  their  power  when  heated  to  100°  C.  In 
1868",  he  reports  that  the  blue  color  is  formed  by  ozone,  pro- 
duced by  a  kind  of  catalytic  action.  He  arrives  at  this  conclu- 
sion after  a  series  of  experiments  with  the  fresh  juice  from 
plants,  and  tincture  of  guaiac,  under  varying  conditions  of  light 
and  air,  and  with  oxygen  obtained  from  other  sources. 

In  1872  Struve23  made  some  experiments  upon  the  change  of 
pyrogallol  to  purpurogalline  by  acacia,  saliva  and  other  substances 
which  produced  a  blue  color  with  tinct.  of  guaiac,  but  did  not 
arrive  at  any  definite  conclusions  as  to  the  true  action. 

In  1877  Traube24  divided  ferments  into  two  groups :    a.  Oxi- 

18  Bulletin  de  Pharmacia  t.  II.  p.  579,  1810. 

18  Journal  de  Fisice  Chim.  etc.  2d.  Semestre,  1819. 

'M  Journal  de  Pharmacie  t.  VI.  1820,  pp.  16-25. 

!1  Jour,  fur  Prakt.  Chem.  67,  1856,  496. 

"-  Jour,  fur  Prakt.  Chem.  105,  1868,  p.  198. 

23  Ann.  d.  Chem.  u.  Pharm.  163,  1872,  p.  160. 

"4  Ber.  d.  Chem.  Ges.,  10,  1877,  p.  1985. 


—  32  — 

dizing  ferments,  those  that  take  up  free  oxygen  and  carry  it  to 
another  substance.  In  this  class  he  places  the  ferments  that  pro- 
duce a  blue  color  with  tincture  of  guaiac,  such  as  that  found  in 
potatoes  and  many  other  plants,  b.  Reducing  ferments ;  those 
that  have  the  power  of  changing  the  combined  oxygen,  produc- 
ing not  only  an  oxidation  product  but  also  a  reduction  product, 
as  alcohol  and  carbonic  acid  from  sugar,  by  the  action  of  yeast. 

In  1882  Clermont  and  Cheutard25  obtained  a  considerable 
quantity  of  pur-purgallin  by  exposing  a  solution  of  pyrogallol 
containing  10%  of  acacia  for  several  weeks  to  the  action  of  air — 
but  failed  to  recognize  the  true  cause  of  the  change  in  color. 

In  1883  H.  Yoshida26  was  the  first  to  discover  that  it  was  an 
enzyme  that  acted  as  an  oxidizing  agent.  He  proved  that  the 
milky  juice  of  Rhus  vernicifera  was  converted  into  a  hard  insolu- 
ble black  varnish  by  the  action  of  a  peculiar  diastatic  substance 
contained  in  the  juice  and  that  the  change  took  place  in  the  pres- 
ence of  moisture,  but  more  rapidly  in  moist  oxygen.  Also  that 
the  change  did  not  take  place  at  all  in  the  presence  of  dry  car- 
bonic acid  gas,  or  in  a  solution  that  had  been  previously  boiled, 
thus  proving  that  the  color  change  was  due  to  a  distase  which 
was  destroyed  by  heat.  He  also  proved  that  the  substance  acted 
upon  by  the  enzyme  had  taken  up  oxygen,  but  did  not  give  off 
carbonic  acid. 

Bouffard27  states  that  the  disease  of  wines  which  causes  a 
skin  to  grow  on  the  surface  of  the  wine  is  due  to  an  enzyme  and 
that  if  the  action  is  allowed  to  continue  the  wine  becomes  color- 
less or  light  yellow.  Later28  he  states  that  this  action  can  be  pre- 
vented by  heating  the  wine  to  60°  or  by  adding  a  very  small 
quantity  of  sulphuric  acid. 

Gouirand29  found  that  by  filtering  diseased  wine  through 
porous  tile  and  precipitating  the  filtrate  with  alcohol  he  obtained 
a  substance  which,  when  added  to  sound  wine,  changed  it  rapidly 
to  the  diseased  condition. 

Martinand30  obtained  a  substance  in  ripe  grapes,  pears  and 


25  Compt.  rend.  97,  1882,  p.  1254. 
20  Jour.  Chem.  Soc.  43,  1883,  p.  472. 

27  Compt.  rend.  118,  1894,  p.  827. 

28  Compt.  rend.  124,  1897,  p.  706. 

29  Compt.  rend.  120,  1895,  p.  887. 

33  Compt.  rend.  121,  1895,  p.  502.     Also  124,  1897,  p.  512. 


33 

apples  which  gives  the  reactions  of  laccase  but  does  not  seem 
to  be  identical  with  it. 

Cazeneuve31  found  that  laccase  produced  only  a  very  slight 
action  in  wines.  He  therefore  attributed  the  disease  to  a  partic- 
ular enzyme  and  named  it  "Oenoxydase."  He  obtained  it  in  the 
form  of  a  gum  by  precipitating  it  from  the  wine  by  the  addition 
of  a  large  amount  of  alcohol.  It  colors  guaiac  blue,  is  active  at 
o°C.  and  is  not  entirely  destroyed  at  65 °C.  He  finds  that  the  col- 
oring matter  of  wines  is  a  phenol-like  body  which  is  oxidized  by 
oenoxydase. 

Laborde32  attributes  the  secretion  of  Oenoxydase  to  a  mould, 
Botrytis  cinerea  (sweet  rot),  which  is  present  at  the  root  of  the 
vine. 

Eduard  SchaY3  has  especially  examined  the  enzyme  in  Phyto- 
lacca  decandra.  He  used  an  extract  prepared  by  macerating  the 
fresh  parts  of  the  plant  in  glycerin  containing  not  more  than  5  or 
10%  of  water,  for  a  few  days,  then  filtering.  He  states  that  an 
extract  so  prepared  scarcely  loses  any  of  its  activity  for  a  year 
and  a  half.  He  found  the  extract  from  the  leaves  to  be  the  most 
active,  that  from  the  root  less  active,  and  that  from  the  flowers 
the  least  active. 

Schar34  states  that  the  blue  color  produced  by  enzymes  upon 
tincture  of  guaiac  depends  upon  the  formation  of  a  peculiar  oxy- 
gen combination  with  the  resinous  constituent  of  guaiac,  the 
guaiaconic  acid.  To  the  blue  compound  he  gives  the  name  ozon- 
ized-guaiaconic  acid.  He  also  states  that  guaiaconic  acid  is  very 
sensitive  to  the  action  of  acids,  alkalies,  light,  air  and  water  and, 
that  when  the  tincture  is  to  be  used  as  a  reagent,  it  should  be 
prepared  fresh,  of  the  strength  of  2  to  3  per  cent,  of  resin  free 
from  wood. 

The  most  valuable  contributions  regarding  the  action  of  sol- 
uble ferments  have  been  given  in  a  series  of  articles  by  G.  Bert- 
rand33.  He  has  given  the  name  "Laccase"  to  the  enzyme  first 
found  in  Japanese  lac  by  Yoshida  but  since  found  in  many  plants. 


81  Compt.  rend.  124,  1897,  p.  406  and  781. 

82  Compt.  rend.  126,  1898,  p.  536. 

33  Vierteljahrsschrift  d.  Naturf.  Ges.  Zurich,  XU   (1896)  233. 

84  66  Versammlung  deutscher  Naturforscher  und  Aertze  &  Wien,  1904. 

85  Bull.  Soc.  3d.  Series  to  51,  p.  159,  1891. 
Compt.  rend.  119,  p.  1012,  1894. 


—  34  — 

Bertrand  named  the  gummy  substance  that  was  separated 
from  Japanese  lac,  "Laccase".  He  has  since  used  a  somewhat 
different  method  of  separation  in  order  to  prepare  it  from  other 
plants,  like  potatoes,  turnips,  beets,  artichokes,  asparagus,  apples, 
pears,  etc.  The  fresh  parts  of  the  plants  are  crushed,  the  juice 
expressed  and  after  saturating  with  chloroform  allowed  to  stand 
24  hours  when  a  coagulum  forms  and  the  juice  is  separated  and 
the  gum  is  then  precipitated  by  alcohol. 

Bourquelot  and  Bertrand36  examined  about  200  species  of 
mushrooms  of  which  they  give  the  following: 

GENERA                                   EXAMINED  ACTIVE           INACTIVE 

Russule  18  18  o 

Lactarius    20  18  2 

Psalliota 5  4  i 

Boletus    18  10  8 

Clitocybe    9  5  4 

Marasmius    6  o  6 

Cortinarius    12  i  n 

Inocybe    6  i  5 

Amanite    7  2  5 

Hygrophorius    6  o  6 

The  parts  of  plants  which  contain  the  least  chlorophyl  con- 
tain the  most  laccase. 

Bertrand37  has  shown  by  experimenting  upon  such  substances 
as  hydroquinone,  pyrogallol,  gallic  acid,  etc.,  that  the  soluble 
ferments  like  laccase  act  by  direct  oxidation  and  that  under  its 
influence  these  bodies,  in  the  presence  of  air,  take  up  oxygen  and 
give  off  carbon  dioxide.  He  found  that  the  phenols  most  easily 
acted  upon  are  those  having  hydroxyl  in  the  ortho  or  para  posi- 
tion. When  in  the  meta  position  they  are  oxidized  with  great 
difficulty. 

On  adding  laccase  to  a  solution  of  hydroquinone  it  changes 
to  a  deep  red  color  and  after  some  time  green  crystals  are  formed 
and  the  solution  has  the  characteristic  odor  of  quinone. 

Bertrand88  states  that  the  darkening  of  certain  substances 
as  the  dahlia,  beet,  etc.,  is  due  to  the  oxidation  of  ty rosin  under 
the  influence  of  soluble  ferments.  But  that  tyrosin  resists  in- 
definitely the  action  of  gaseous  oxygen  in  the  presence  of  laccase, 
even  in  strong  solutions  ;therefore  the  blackening  of  the  tyrosin 


30  Compt.  rend.  121,  p.  166  &  783,  1895. 

37  Compt.  rend.  120,  p.  226,  1895. 

38  Compt.  rend.  122,  p.  1215,  1896. 


—  35  — 

in  the  dahlia  and  beet  is  due  to  a  peculiar  oxidizer.  This  he  has 
been  able  to  separate  and  has  called  it  "Tyrosinase".  It  exists 
not  only  in  the  dahlia  and  beet  but  in  several  varieties  of  mush- 
rooms which  do  not  contain  ty  rosin. 

Tyrosinase  is  very  unstable.  It  is  best  prepared  from  Rus- 
sules. 

One  can  either  use  juice  of  the  mushrooms  at  once,  or  pre- 
serve them  for  future  use  by  cutting  in  thin  slices  and  drying  in 
a  vacuum.  When  wanted  for  use  the  dried  residue  is  macerated 
for  some  time  in  cold  water  and  then  filtered. 

If  a  solution  of  tyrosinase  is  mixed  with  a  solution  of  ty- 
rosin  and  the  liquid  frequently  shaken  to  introduce  air  the  liquid 
will  first  turn  red,  then  black.  Tyrosinase  is  not  so  frequently 
found  in  plants  as  laccase,  but  may  be  found  in  many  fungi  such 
as  Russula,  Lactarius,  Hebeloma,  Boletus,  Amonita,  and  many 
others. 

The  two  enzymes  frequently  exist  in  the  same  plants.  Bert- 
rand  found  that  tyrosinase  was  more  easily  destroyed  by  heat 
than  laccase. 

Bertrand39  gives  the  following  as  the  theory  of  the  action  of 
oxydizing  enzymes.  That  the  manganese,  which  is  present  in  all 
enzymes,  even  as  high  as  2%,  exists  in  the  albuminous  substance 
as  a  manganous  compound,  and  plays  the  part  of  oxygen  carrier. 
The  oxygen  molecule  is  broken  by  the  manganous  compound  to 
form  manganese  dioxide  and  the  remainder  of  the  oxygen  mole- 
cule acts  upon  the  oxydizible  body  present.  Finally  through  the 
acid  character  of  the  albumen  radical,  the  manganese  dioxide  is 
decomposed  and  the  original  manganese  compound  restored.  He 
believes  that  the  activity  of  the  enzyme  is  proportional  to  the 
manganese  present. 

Bourquelot40  reports  upon  the  action  of  tyrosinase  on  phenols, 
etc.  He  states  that  the  tyrosinase  of  mushrooms  forms  with  gua- 
iacol  a  red  precipitate. 

Bokorny41  after  giving  the  apparent  similarity  of  enzymes 
and  protoplasm,  especially  with  reference  to  the  action  of  light 
and  temperature,  concludes  that  one  can  scarcely  think  that  they 
originate  from  the  same  source. 


39  Compt.  rend.  124,  1356,  1897. 

40  Repertoire  de  Pharmacie,  1897,  p.  136. 

41  Allgeminen  Brauer  und  Hopfen  Zeitung,  No.  74,  1901. 


-36- 

Loew42  reports  two  kinds  of  enzymes,  an  insoluble  and  a 
soluble  form — a-  and  ^-catalase  respectively.  The  former  is  prob- 
ably a  compound  of  the  soluble  catalase  with  a  nucleoproteid. 
while  the  soluble  catalase  is  an  albumose  and  can  be  liberated  by 
the  action  of  very  dilute  alkaline  media  upon  the  insoluble  cata- 
lase. He  has  studied  these  enzymes  with  special  reference  to  the 
tobacco  plant. 

Catalase  does  not  color  tincture  of  guaiac  blue,  but  changes 
hydroquinone  into  quinone.  Traces  of  acids  increases  its  action 
while  alkalies  destroy  it. 

Chodat  and  Bach43  state  that  catalase  is  not  a  true  enzyme 
like  oxydase  or  peroxydase,  as  its  function  is  only  to  decompose 
hydrogen  peroxide. 

Kastle  and  Shedd44  have  shown  that  phenolphthalin  is  oxi- 
dized to  phenolphthalein  by  oxidizing  ferments  and  that  it  forms 
a  very  sensitive  reagent  for  the  presence  of  soluble  enzymes. 
They  have  tested  this  reagent  upon  a  number  of  enzymes  from 
plants  and  on  a  few  from  animals.  These  results  were  com- 
pared with  those  obtained  by  tincture  of  guaiac  and  found  to  be 
practically  identical,  i.  e.,  all  those  enzymes  which  gave  a  blue 
color  with  guaiac,  gave  a  pink  color  with  phenolphthalin,  the 
colors  in  both  cases  increasing  or  decreasing  with  the  activity 
of  the  enzyme. 

In  i8c)845  L,aborde  proposed  to  measure  the  activity  of  en- 
zymes by  comparing  the  color  produced  by  the  enzyme  when  act- 
ing upon  an  alcoholic  tincture  of  guaiac,  with  a  standard  color 
formed  by  adding  0.5  gramme  of  iodine  to  20  cc.  of  the  tincture 
of  guaiac. 

Alliot  and  Pozzi-Escot48  found  it  impossible  to  estimate  oxy- 
dases  colorometrically  either  by  Laborde's  guaiac  method  or  by 
Kastle  and  Shed's  phenolphthalein  method. 

Kastle  and  Shed  found  that  the  only  enzyme  obtained  from 
animal  source,  which  acted  as  an  oxidizing  enzyme  was  the 
human  saliva. 

About  this  time  Cavazzani47  found  a  soluble  oxidizing  en- 


42  U.  S.  Dept.  of  Agriculture,  Report  No.  68,  1901. 


M 

Am.  Chem.  Jour.  26,  1901,  p.  526. 
45  Compt.  rend.  126,  1898,  p.  536. 
**  Ann.  Chem.  Anal.  7,  1902,  p.  210. 
41  Cent.  Physiol.  14,  1901,  p.  473. 


—  37  — 

zyme  in  the  cerebro-spinal  fluid  of  dogs  and  calves,  and  Vitali48 
reported  oxydase  in  pus,  which  he  obtained  by  triturating  the  pus 
with  glass,  and  then  extracting  with  water,  dilute  acetic  acid,  or 
with  equal  parts  of  glycerin  and  water.  It  imparted  a  blue  color 
to  tincture  of  guaiac.  This  action  was  destroyed  by  hydrocyanic 
acid,  chloroform,  hydroquinone,  pyrogallol,  and  hydroxylamine, 
but  not  by  phenol,  thymol  or  mercuric  chloride. 

Gersard49  found  in  the  ink  sac  of  the  cuttle-fish  laccase,  tyros- 
inase  and  an  oxidizing  diastase  which  is  more  resistant  to  heat 
that  laccase. 

Kastle  and  Loevenhart50  while  studying  the  action  of  oxidiz- 
ing enzymes  have  established  the  fact  that  the  organic  peroxides 
like  benzol,  phthalyl  and  succinyl  peroxides  will  color  tincture  of 
guaiac  blue. 

The  same  authors  have  very  thoroughly  studied  the  poison- 
ous action  of  a  variety  of  substances  upon  enzymes,  and  also  the 
effect  of  the  same  substance  upon  organic  peroxides,  and  find 
that  those  substances  which  destroyed  the  power  of  enzymes 
to  color  guaiac  blue  also  prevented  the  coloration  by  organic 
peroxides. 

They  made  many  experiments  with  the  juice  from  the  potato 
which  colored  guaiac  blue,  and  oxidized  phenolphthalin,  but  the 
enzyme  could  not  be  precipitated  by  absolute  alcohol  as  most  en- 
zymes. They  arrive  at  the  following  conclusions: 

"i.     That  oxygen  is  absolutely  essential  to  the  production  of 
the  guaiacum-bluing  ferment  of  the  potato. 

"2.  That  this  so-called  oxidizing  ferment  is  in  all  probability 
not  a  true  ferment,  but  an  organic  peroxide. 

"3.  That  the  oxidation  phenomena  occurring  in  the  plant, 
and  probably  in  the  animal  organism  also,  can  be  satisfactorily 
explained  upon  the  supposition  that  the  readily  autoxidizable 
substance  which  they  contain  is  oxidized  to  the  peroxide  condi- 
tion by  molecular  oxygen,  and  that  the  peroxides  thus  formed 
in  turn  give  up  part  of  their  oxygen  to  other  less  oxidizable  sub- 
stances present  in  the  cell.  In  other  words,  that  the  process  of 
rendering  oxygen  active,  by  the  living  cell,  is  probably  brought 


4S  L'Orosi,  24,  1901,  p.  263,  from  Jour.  Chem.  800.4.  ii,  672. 
4J  Compt.  rend.  136,  1903,  p.  631. 
60  Am.  Chem.  Jour.  26,  1901,  p.  539. 


-38- 

about  in  essentially  the  same  way  that  is  accomplished  by  phos- 
phorus, benzaldehyde  and  other  oxygen  carriers." 

Hunger51  states  that  the  guaiac  reaction  is  interfered  with 
by  tannins,  certain  sugars,  hydrogen  sulphide,  pyrogallol  and 
other  reducing  agents. 

Pozzi-Escof'2  reports  that  when  living  tissues  of  animal  or 
vegetable  origin  do  not  affect  guaiac,  but  decompose  hydrogen 
peroxide,  that  reductase  must  be  looked  for,  which  may  be  done 
by  treating  them,  out  of  contact  with  air,  with  a  solution  of  in- 
digo, or  litmus,  or  ferric  ferricyanide  and  note  if  any  reduction 
has  taken  place.  They  also  liberate  hydrogen  sulphide  from  a 
mixture  of  sulphur  and  potassium  fluoride,  if  protected  from  the 
air. 

Bach  and  Chodat53  find  that  oxydase  is  always  accompanied 
by  peroxydase,  which  they  have  found  in  about  25  plant  families. 
The  oxydase  has  the  power  of  forming  peroxides  in  the  presence 
of  free  oxygen,  which  can  be  detected  by  the  liberation  of  iodine 
from  hydriodic  acid.  When  parts  of  plants  containing  these  en- 
zymes are  heated  to  80°  C.  the  power  to  liberate  iodine  or  color 
guaiac  blue  is  destroyed.  This  is  also  true  of  the  expressed  juice. 
The  power  to  liberate  iodine  disappears  more  quickly  than  that 
which  colors  guaiac.  When  the  substance  ceases  to  act  it  may 
be  made  active  again  by  the  addition  of  a  small  quantity  of  hydro- 
gen peroxide. 

Bach54  showed  that  a  large  number  of  substances  when  sub- 
mitted to  slow  oxidation  in  the  air,  formed  peroxides,  and  that 
the  peroxide  formed  helped  to  continue  the  oxidation.  In  the 
blood  the  easily  oxidizable  substances  first  form  peroxides  and 
these  aid  in  the  oxidation  of  the  more  difficultly  oxidisable  bodies. 
The  oxidation  does  not  seem  to  be  influenced  by  light. 

Wender55  thought  that  the  action  of  the  oxydase  in  the  living 
cell  is  to  cause  the  oxygen  of  the  aeroxydase  to  oxidize  the  easily 
oxidizable  bodies  so  that  intermediate  bodies  are  formed,  then 
these  are  again  decomposed  by  the  action  of  catalase.  The  free 
oxygen  by  the  action  of  anaeroxydase  (peroxides)  becomes  active 
and  oxidizes  difficultly  oxidizable  substances. 


51  Ber.  d.  Bot.  Ges.  19,  1901,  p.  648. 
62  Ann.  Chem.  anal.  7,  1902,  260. 

53  Ber.  d.  Chem.  35,  1902,  p.  2466. 

54  Compt.  rend.  124,  1897,  p.  951. 

55  Chem.  Zeit.  26,  p.  1217  &  1221,  1902. 


—  39  — 

Chodat  and  Bach56  prepared  peroxydase  from  cucumbers  and 
horseradish  but  on  account  of  the  large  amount  of  water  in  the 
former,  they  preferred  to  prepare  it  from  the  horseradish  as  the 
yield  was  much  larger.  Their  method  was  to  reduce  it  to  as  fine 
a  condition  as  possible  and  allow  it  to  stand  for  an  hour  to  permit 
the  glucoside-splitting  enzyme  to  act,  then  to  express  the  juice 
and  precipitate  with  absolute  alcohol.  The  precipitate  was  ex- 
tracted with  40%  alcohol,  the  alcoholic  solution  concentrated  in 
a  vacuum  at  30°  C.  and  precipitated  with  absolute  alcohol  and 
dried  in  a  vacuum. 

The  product  is  a  yellowish  gummy  mass  which  in  solution 
reduces  Fehling's  solution ;  however  they  claim  that  this  is  not 
due  to  the  enzyme,  for  by  repeated  precipitation  an  enzyme  may 
be  obtained  which  will  not  reduce  Fehling's  solution. 
The  purest  peroxydase  obtained  from  horseradish  con- 
tained 6  per  cent,  of  ash  of  which  aluminum  amounted 
to  0.8  to  1.4  per  cent,  and  manganese  from  0.2  to  0.6  per  cent. 
They  found  that  the  peroxydase  from  Russule  and  Lactar- 
ius  was  much  more  active  than  that  from  the  horseradish  or 
cucumber ;  on  the  other  hand  with  hydrogen  peroxide  the  activity 
was  reversed.  By  heating  a  mixture  of  oxydase  and  peroxydase 
to  70°  C.  the  former  is  destroyed  while  the  latter  is  only  weaken- 
ed but  seems  to  regain  its  strength  on  standing"  but  if  subjected 
to  a  second  heating  its  activity  is  destroyed  completely.  In  alco- 
holic solution  it  is  destroyed  by  boiling.  Peroxydase  does  not 
possess  the  power  to  oxidize  except  in  the  presence  of  peroxides. 
The  same  authors  state08  that  peroxydase  and  catalase 
are  present  in  nearly  all  parts  of  plant  and  animal 
bodies,  and  apparently  are  antagonistic,  as  the  first  is 
active  with  hydrogen  peroxide  while  the  other  is  destroyed 
by  oxygen.  They  experimented  upon  a  mixture  of  oxygenase, 
peroxydase  and  pyrogallol  alone,  and  in  the  presence  of  catalase, 
and  found  that  catalase  had  no  effect  upon  the  amount  of  oxygen 
absorbed.  In  another  experiment  catalase  was  mixed  with  oxy- 
genase and  peroxydase,  and  the  mixture  allowed  to  stand  over 
night,  then  hydrogen  peroxide  was  added  and  the  amount  of  gas 
liberated  was  found  to  be  the  same  as  without  the  catalase.  Hence 


M  Ber.  d.  Chem.  36,  1903,  p.  600. 

57  Compare  U.  S.  Dept.  of  Agric.,  Bull.  No.  8,  17. 

BS  Ber.  d,  Chem.  36,  1903,  p.  1756. 


—  40  — 

they  conclude  that  peroxydase  and  catalase  can  exist  in  the  same 
plants  without  interfering  with  the  functions  of  either.  They 
also  prove  that  catalase  and  reductase  are  not  identical. 

Kunz-Krause59  and  Dr.  Richard  Fibras60  believe  that  one  of 
the  causes  of  the  deposit  in  tinctures  is  due  to  the  action  of  en- 
zymes. 

Bourquelot81  studied  the  ferments  which  cause  hydrolisis 
of  the  various  polysaccharides,  and  finds  that  two  and  in  some 
cases  three  ferments  are  required  to  completely  hydrolise  poly- 
saccharides. In  another  paper,  with  Herissey62  he  deals  with  the 
ferment  of  milk,  almonds,  peach  and  cherry  laurel  leaves  and  finds 
that  emulsine  as  obtained  from  almonds,  is  a  mixture  of  several 
ferments,  emulsin,  lactase  and  probably  gentiobias,  and  frequently 
invertin.  He  concludes: 

1.  That  lactase  accompanies  emulsin  in  the  different  al- 
monds of  rosaceae. 

2.  That  emulsin   exists   without  laccase,   as   in   Perquillus 
niger  leaves. 

3.  That  lactase  exists  without  emulsin,  as  in  the  yeast  of 
kaphis. 

Chodat  and  Bach63  review  the  researches  of  soluble  ferments 
(oxydase)  and  give  the  following  theory:  The  oxidizing  fer- 
ments called  oxydase,  are  bodies  of  a  peroxide  character,  there- 
fore they  are  organic  peroxides  which  heat  decomposes.  Their 
action  is  accelerated  by  a  second  class  of  bodies  which  act  as 
catalytics,  and  have  the  power  to  bring  back  the  peroxides  to 
their  normal  condition.  They  state  that  there  is  present  in  the 
living  cell  a  diastase  (peroxydase)  which  acts  on  hydrogen  per- 
oxide similar  to  ferrous  sulphate,  and  with  hydrogen  peroxide 
produces  a  blue  color  with  guaiac. 

Chodat  and  Bach  give  the  following  characteristics  for  per- 
oxydase :M 

If  free  from  other  enzymes,  it  does  not  liberate  oxygen  from 


59  Pharm.  Centralhalle,  1902,  No.  52. 

90  Pharm.  Post,  35,  1902,  p.  548. 

81  Jour.  d.  Chim.  et  d.  Pharm.,  1903. 

Also  Compt.  rend.  136,  p.  762,  1903. 
62  Jour.  d.  Chim.  et  d.  Pharm.,  1903. 

Also  Compt.  rend.  Soc.  Biol.  55,  p.  219,  1903. 
"  Ber.  d.  Chem.  35,  P-  1275,  1902. 
°*  Ber.  d.  Chem.  37,  p.  1342,  1904- 


—  41  — 

hydrogen  peroxide  as  Catalase,  or  oxydize  pyrogallol  as  Oxy- 
genase ;  or  liquefy  starch  paste,  forming  substances  which  de- 
duce Fehling's  solution,  as  Amylase;  or  invert  cane  sugar,  as 
Invertase ;  or  break  up  glucosides,  as  Emulsin ;  or  digest  coagu- 
lated albumen,  as  the  Proteolytic  enzymes. 

Chodat  and  Bach"  state  that  Bertrand  found  that,  by  frac- 
tional precipitation  of  laccase  with  alcohol,  he  obtained  a 
substance  poor  in  manganese  and  weak  in  oxidizing  power,  also 
a  substance  rich  in  manganese  and  strong  in  oxidizing  action, 
but  did  not  suspect  that  the  decrease  in  power  of  oxidation  was 
connected  with  the  separation  of  peroxydase.  Five  years  later 
fractional  precipitation  was  brought  forward  as  a  method  of  sepa- 
ration of  peroxydase  from  oxydase,  by  Aso.M 

The  authors  have  used  this  method  to  obtain  two  end  frac- 
tions, one  with  weak  oxidizing  power  and  the  other  without  any 
oxidizing  action.  The  first  was  practically  insoluble  in  40%  alco- 
hol while  the  other  was  soluble,  and  was  active  with  hydrogen 
peroxide,  and  behaved  as  a  true  peroxydase.  The  weak  oxidizing 
fraction  which  principally  took  the  part  of  an  oxygen  carrier 
they  designate  as  Oxygenase.  They  add  that  it  is  comparatively 
easy  to  prepare  peroxydase  free  from  oxygenase,  but  have  not 
succeeded  in  preparing  oxygenase  free  from  peroxydase.  A  par- 
tial separation  may  be  made  by  extracting  a  mixture  of  the  two 
with  30  to  60  per  cent,  alcohol,  or  by  dialysis  with  pure  water 
when  the  peroxydase  passes  into  the  dialysate.67 

In  1892  Tschirch68  called  attention  to  the  fact  that  the  differ- 
ence between  the  color  of  black  and  green  tea  was  due  to  fer- 
mentation. The  black  tea  is  prepared  by  allowing  the  fresh  leaves 
to  undergo  partial  fermentation  at  a  relatively  low  temperature, 
while  in  the  case  of  green  tea  the  ferment  is  destroyed  by  heat. 

Aso69  states  that  the  color  of  black  tea  is  produced  by  the 
action  of  oxydase  on  the  tannin  in  the  first  stage  of  preparation, 
while  in  the  green  tea  the  ferment  is  destroyed. 

Tschirch  and  Oesterle70  state  that  the  formation  of  cola-red 


co  Ber.  d.  Chem.  36,  606,  1903. 
50  Bull.  Coll.  Agric.  Tokio,  5,  2,  1902,  p.  233. 

67  Compare  Engler  and  Wild,  Ueber  die  sogenannte  activirung  des 
Sanerstofrs  und  iiber  Superoxyd  bildung.     Ber.  d.  Chem.  30,   1669,   1897. 
m  Indische   Heil-und   Nutzpflanzen   und   deren   Kultur,   Berlin,    1892. 
"  Bull.  Coll.  Agric.,  Tokio,  4,  p.  254,  1901. 
"  Anatom.  Atlas  d.  Pharmacognosie,  p.  350. 


—  42  — 

in  cola  nuts  is  produced  by  fermentation,  and  that  the  nuts  may 
be  preserved  colorless  by  heating  to  65  degrees,  or  by  immersing 
in  boiling  alcohol. 

Tschirch71  observed  that  cinchona  bark  does  not  become  col- 
ored, if  the  fresh  branches  are  immersed  in  hot  water  before  re- 
moving the  bark. 

The  same  author  states  that  it  is  the  action  of  oxydases  upon 
the  tannin  group,  which  produces  the  strong  red-brown  products 
that  he  has  grouped  together  as,  cinchona-red,  tannin-red,  cinna- 
mon-red, kino-red,  colo-red,  etc.7" 

Wender73  attributes  the  brown  color  of  bread  to  the  action 
of  an  enzyme,  and  adds  that  the  brown  color  of  many  trees  is 
due  to  the  action  of  enzyme  on  the  tannin. 

Browne74  found  lipase  in  rice  bran  and  tested  its  hydrolytic 
action  on  castor  oil  and  upon  the  oil  from  rice  bran. 

Mohr75  finds  that  lipase  acts  as  a  hydrolizer  in  the  decompo- 
sition of  esters  but  that  the  decomposition  is  not  complete  if  only 
alcohol  and  acid  are  present.  After  a  time  the  action  is  reversed 
and  esters  are  formed  from  the  acid  and  alcohol  present. 

Wender  and  Lewin76  state  that  the  enzyme  of  grain  which  has 
a  catalytic  action  is  not  increased  during  germination.  Also  that 
the  diastatic  action  may  be  destroyed  by  carefully  increasing  the 
heat  , without  destroying  the  catalytic  action.  Also  that  the  outer 
thin  brown  seed  coat  contains  the  strongest  enzyme,  and  decreases 
in  strength  toward  the  center. 

Bourquelot  and  Marchadier77  find  that  oxydase  and  peroxy- 
dase  are  active  in  10%  alcohol,  and  that  both  are  destroyed  by 
hydrocyanic  acid.  With  vanilla,  peroxydase  and  hydrogen  per- 
oxide act  the  same  as  oxydase  with  air,  and  suggest  that  per- 
oxydase consists  of  two  enzymes,  one  hydroperoxydase  which, 
in  the  presence  of  air,  is  capable  of  converting  water  into  hydro- 
gen peroxyde,  or  forming  peroxides  with  certain  substances,  and 
the  other  an  indirect  oxydase,  capable  of  decomposing  the  per- 
oxides with  the  liberation  of  oxygen. 


71  Schweiz.  Wochschr.  fiir  Pharm.,  No.  10,  1905. 

72  Angew.  Pflanzenanatomie,  p.  127. 

73  Chem.  Zeit,  26,  p.  1217,  1902. 

74  Jour.  Am.  Chem.  Soc.,  25,  p.  950,  1903. 

75  Chem.  Centr.,  1902,  ii,  1424,  from  Woch.  Braii.   19,  p.  588. 

78  Chem.  Centr.,  1904,  I,  p.  1530,  from  Oest.  Chem.  Zeit.,  7,  p.  173,  1904. 
77  Compt.  rend.  138,  p.  1432,  1904. 


^V: 

f  UNIVERSITY  ) 

-43-  ^^       ^  J 

^^c. '. ; 

GUM    AND   ENZYME. 

After  extracting  the  lac  with  alcohol  the  residue  was  extract- 
ed with  cold  water  and  the  gum-enzyme  precipitated  by  pouring 
into  strong  alcohol.  The  precipitate  was  dissolved  in  a  small 
quantity  of  water  and  reprecipitated  with  alcohol.  By  repeating 
the  operation  several  times  and  finally  washing  with  ether  and 
drying  in  an  exsiccator,  it  was  obtained  perfectly  white  and  easily 
reduced  to  powder.  In  physical  appearance  it  is  similar  to  pow- 
dered acacia.  When  so  prepared  the  gum-enzyme  is  very  active, 
rapidly  changing  tincture  of  guaiac  to  a  deep  blue  color.  If 
an  emulsion  is  made  with  the  gum-enzyme,  water  and  the  sepa- 
rated resin,  it  soon  changes  from  yellowish-white  to  black.  A 
solution  of  gum-enzyme  with  naphtol  formed  a  purplish  blue 
color,  and  with  guiacol  a  red  color  in  half  an  hour  but  produced 
no  effect  upon  a  solution  of  vanillin  in  hydrochloric  acid.  If  a 
solution  of  the  gum-enzyme  is  boiled  with  water,  it  becomes  en- 
tirely inactive. 

TESTS  FOR  NITROGEN. 

It  is  a  generally  conceded  fact  that  all  enzymes  contain  nitro- 
gen. The  Lassaigne  test  for  the  detection  of  nitrogen  is  un- 
doubtedly considered  the  most  reliable.  It  consists  in  heating  the 
substance  with  metallic  potassium  or  sodium  and  converting  the 
cyanide  so  formed  into  Prussian  blue.  This  test  was  applied  to 
the  gum-enzyme,  but  failed  to  detect  the  presence  of  nitrogen. 
According  to  Kehrer  the  Lassaigne  test  must  be  modified  for 
certain  pyrrol  derivatives,78  and  cannot  be  applied  to  diazocom- 
pounds.79  In  view  of  the  certainty  of  the  presence  of  nitrogen 
and  the  general  reputation  of  the  test,  it  was  repeatedly  tried  with 
various  modifications.  The  gum-enzyme  was  previously  mixed 
with  dry  sodium  carbonate  and  carefully  ignited.  The  rapidity 
of  the  heating  was  varied.  In  another  experiment  the  sub- 
stance was  placed  in  a  narrow  tube  closed  at  one  .end,  and  the 
tube  drawn  out  to  contract  the  opening,  small  pieces  of  sodium 
were  then  introduced  and  the  tube  again  contracted,  thus : 


c 


GUM-ENZYME  SODIUM 


78  Berichte,  35,  2,525 ;  1902. 

79  Berichte,  17,  1,178;  1884. 


—  44  — 

The  sodium  was  first  heated,  then  the  gum-enzyme  slowly  heated 
so  that  the  gases  would  pass  over  the  glowing  sodium.  This  test 
was  repeated  in  the  same  manner,  except  that  the  gum-enzyme 
was  first  mixed  with  dry  potassium  hydroxide.  In  another  ex- 
periment the  substance  was  heated  with  a  small  quantity  of  con- 
centrated sulphuric  acid  until  a  dry  charred  mass  was  obtained, 
then  mixed  with  metallic  iron  and  sodium  and  ignited,  and  fin- 
ally tested  for  cyanide.  In  another  experiment  a  modification  of 
the  Kjeldahl  quantitative  method  was  tried.  The  gum-enzyme 
was  heated  with  concentrated  sulphuric  acid  and  a  little  mercuric 
oxide  until  a  colorless  solution  was  obtained.  The  solution  was 
then  mixed  with  an  excess  of  potassium  hydroxide  and  distilled. 
The  distillate  was  passed  through  a  tube  containing  a  piece  of 
red  litmus  paper  into  a  mixture  of  chloroform,  alcohol  and  po- 
tassium hydroxide  to  convert  the  ammonia  into  cyanide.  The 
litmus  paper  remained  red  throughout  the  distillation.  All  at- 
tempts to  convert  the  nitrogen  into  cyanide  failed. 

Another  test  for  nitrogen  which  is  considered  less  reliable 
than  the  Lassaigne  test,  is  to  convert  the  nitrogen  into  ammonia 
by  heating  the  substance  in  a  tube  with  soda-lime  or  potassium 
hydroxide.  This  test  was  applied  to  the  gum-enzyme  when  the 
red  litmus  paper  placed  over  the  end  of  the  tube  rapidly  changed 
to  blue,  but  no  odor  of  ammonia  could  be  detected.  The  paper 
was  evenly  colored  as  if  produced  by  some  gaseous  substance. 
The  test  was  repeated  with  a  pledget  of  cotton  inserted  in  the 
tube  below  the  paper  to  prevent  the  possibility  of  potassium  hy- 
droxide being  mechanically  carried  to  the  litmus  paper.  The  re- 
sult was  the  same  as  in  the  previous  test.  A  blank  test  was  next 
made  under  exactly  the  same  conditions,  but  with  negative  re- 
sults. These  experiments  indicated  the  presence  of  a  volatile 
base.  Professor  Tschirch  thought  the  odor  similar  to  pyrrol.  I, 
therefore,  repeated  the  test,  placing  in  the  top  of  the  tube  a  pine 
shaving  moistened  with  hydrochloric  acid.  This  was  rapidly 
colored  red,  thus  strongly  indicating,  if  not  conclusively  proving, 
the  presence  of  pyrrol,  or  a  pyrrol  derivative.  This  was  further 
confirmed  by  placing  5  grammes  each  of  powdered  potassium 
hydroxide  and  gum-enzyme  in  a  flask  and  distilling.  The 
vapors  were  passed  through  a  condenser  connected  with  a  dry 
flask,  and  this  again  connected  with  a  second  flask  by  means  of 
a  tube  which  passed  to  the  bottom  of  the  flask,  into  a  small  quan- 
tity of  water.  At  the  end  of  the  reaction  the  first  flask  contained 


—  45  — 

a  small  quantity  of  colorless,  strongly  alkaline  liquid,  sparingly 
soluble  in  water,  but  readily  soluble  in  alcohol  and  ether.  The 
solution  was  tested  with  the  following  results: 

On  warming  with  hydrochloric  acid  and  allowing  to  stand 
a  short  time  a  fine  red  precipitate  separated.  With  sulphuric 
acid  and  quinone  a  green  precipitate  formed ;  with  phosphomo- 
lydic  acid,  first  a  yellow,  then  a  blue  precipitate ;  with  potassium 
ferrocyanide,  dark  green ;  with  quinone  alone,  violet  red.  The 
contents  of  the  second  flask  was  also  alkaline.  The  distillate 
was  also  tested  by  the  Lassaigne  test,  but  no  Prussian  blue  ob- 
tained. As  pyrrol  gives  the  Lassaigne  test  it  must  be  that  the 
distillate  did  not  consist  of  pyrrol,  but  was  a  pyrrol  derivative. 

Another  evidence  of  the  presence  of  nitrogen  was  obtained 
as  follows :  An  ordinary  open  combustion  tube  was  filled  with 
copper  oxide  and  ignited  in  a  current  of  oxygen.  After  partially 
cooling,  a  platinum  boat  containing  the  gum  was  introduced 
and  the  gum  burned  in  a  current  of  oxygen.  The  products 
of  combustion  were  conducted  through  potash  bulbs  containing 
a  solution  of  potassium  hydroxide,  prepared  from  metallic  po- 
tassium, and  water  distilled  with  potassium  permanganate.  Just 
before  the  combustion  the  solution  was  tested  and  found  to  be 
free  from  nitrogen  compounds.  After  the  combustion  the  solu- 
tion was  tested  with  diphenylamine  when  it  gave  the  blue  color 
characteristic  of  nitrates.  With  brucine  and  sulphuric  acid  a  red 
color  and  with  sulphuric  acid  and  sulphate  of  iron  the  brown  ring 
appeared. 

This  proves  conclusively  that  the  gum  contained  nitrogen  in 
some  form,  which  is  converted  into  pyrrol,  or  a  pyrrol  derivative, 
by  heating  with  potassium  hydroxide. 

ATTEMPTS  TO  SEPARATE  THE  GUM   FROM  THE  ENZYME. 

Hikorokuro  Yoshida  states  that  by  removing  his  so-called 
urushic  acid  with  alcohol  and  extracting  the  residue  with  cold 
water,  and  then  boiling  the  solution,  a  white  precipitate  is  formed. 
He  assumes  that  it  is  the  enzyme,  but  does  not  prove  it,  except 
that  the  solution  was  active  before  boiling  and  inactive  after- 
wards, and  that  the  precipitate  contained  nitrogen.  It  may  have 
been  an  inactive  vegetable  albumen,  although  he  states  that  it 
contained  less  nitrogen  than  these  bodies  usually  contain.  I  have 
found,  however,  that  a  solution  of  the  purified  gum  obtained  by 


-46- 

repeated  precipitation  with  alcohol  remained  perfectly  clear  on 
boiling;  yet,  previous  to  boiling,  the  same  solution  was  strongly 
active,  rapidly  changing  tincture  of  guaiac  to  dark  blue,  and  the 
clear  brown  resin  from  the  lac  to  a  hard,  black,  insoluble  sub- 
stance. ^ 

Solutions  of  the  gum  were  treated  with  acetic,  hydrochloric, 
nitric  and  sulphuric  acids  of  various  strengths  and  with  varying 
degrees  of  heat,  but  each  failed  to  separate  the  nitrogenous  sub- 
stance from  the  gum.  In  one  experiment  the  solution  was  boiled 
for  half  an  hour  with  a  dilute  sulphuric  acid,  precipitated  with 
alcohol,  dissolved  in  water  and  reprecipitated  with  alcohol, 
washed  until  free  from  sulphuric  acid,  and  dried  in  an 
exsiccator.  This  still  gave  the  pyrrol  reaction.  Fractional  pre- 
cipitation was  tried  without  apparent  change  in  the  relation  of 
gum  to  nitrogen.  Cold  saturated  solutions  of  magnesium  sul- 
phate, ammonium  sulphate  and  sodium  phosphate  were  tried  in 
vain.  Various  modifications  of  Almen's  solution  of  tannic  acid 
were  tried,  but  in  no  case  was  there  any  separation  of  nitrogenous 
from  non-nitrogenous  substance.  Numerous  precipitates  were 
obtained,  but  in  every  case  the  precipitate  contained  both  gum 
and  nitrogen  in  apparently  the  same  proportion  as  before.  The 
dry  powdered  gum  was  heated  for  two  hours  at  temperatures 
varying  from  100°  to  160°  C.  and  tested  both  by  boiling  alone 
and  with  acids,  but  no  separation  occurred. 

EXAMINATION   OF   OTHER   GUMS   FOR    NITROGEN. 

A  number  of  the  following  samples  were  prepared  by  stu- 
dents and  kindly  furnished  by  Professor  Tschirch  from  his  col- 
lection. The  remainder  were  prepared  by  the  writer.  In  the  case 
of  the  gum-resins  the  resin  was  removed  by  extracting  with  alco- 
hol, the  gum  dissolved  in  water  and  precipitated  by  alcohol,  puri- 
fied by  repeated  precipitation  and  dried  in  an  exsiccator.  The 
acids  were  prepared  by  the  same  method,  with  the  exception  that 
the  solutions  were  acidulated  with  hydrochloric  acid  each  time 
before  precipitation,  and  the  precipitate  finally  washed  with  alco- 
hol until  free  from  hydrochloric  acid.  Nos.  14  and  15  were  pre- 
pared by  dissolving  the  tragacanth  in  a  warm  solution  of  sodium 
hydroxide,  precipitating  with  alcohol  and  dissolving  in  water, 
and  reprecipitating  with  acidulated  alcohol. 

Each  sample  prepared  without  heat  was  tested  for  enzyme, 


—  47  — 

and  all  were  tested  by  heating  with  potassium  hydroxide  and 
testing  the  vapor  for  alkalinity  and  by  the  pyrrol  reaction.  The 
enzyme's  activity  is  indicated  by  the  time  required  from  the  addi- 
tion of  the  tincture  of  guaiac  to  the  first  appearance  of  color  and 
afterwards  to  time  required  to  produce  a  given  shade.  In  each 
case  o.i  gramme  of  the  gum  was  dissolved  in  4  cc.  of  water  and 
three  drops  of  tincture  of  guaiac  added. 


NO.                   GUM   FROM 

PREPARED  BY 

PYRROL 
REACTION 

LITMUS 

ENZYME 

i    Japanese  Lac  .  

Stevens 

Oscar  Halbey 

Knitl 
Dr.  Saal 
Bergniann 
Stevens 
Schereschewski 
Stevens 

Halbey 
Knitl 
Stevens 

Very  strong 
Weak 
Medium 

Strong 
Weak 

Strong 

Weak 
Medium 

Medium 
Weak 
Medium 
Strong 

B 

ue 

Immediately 
30-  60  Minutes 
15-  60 
8-  13 
I-    4 
I-    4 
60-120 
60- 
30- 
30  Sec.  12  Min. 
15  Minutes 
15 
Inactive 

3    Ammoniac  select 

4    Acacia 

5    Asafoetida 

6    Asafoetida  select  
7    Olibanum_        

8    Olibanum  
9    Opoponax  
10    Tacamohaca  

ii     Myrrh 

12    Galbanum  „  __ 

13    Chicle 

14    Tragacanth,  white  

15    Tragacanth  yellow 

16    Tragacanth,  white  

ACIDS  PREPARED  FROM 

18    Opoponax  

19    Acacia  __ 

20    Asafoetida  

21    Japanese  lac  

Nos.  8,  9,  ii  and  12  did  not  become  as  dark  as  standard, 
even  after  standing  twenty-four  hours.  Heat  was  used  in  the 
manufacture  of  No.  13,  which  would  have  destroyed  the  enzyme 
had  it  been  present. 

As  the  acids  prepared  from  active  gums  did  not  give  the  en- 
zyme reaction,  it  is  evident  that  the  hydrochloric  acid  used  in 
their  preparation  destroyed  the  enzyme,  but  did  not  remove  the 
nitrogen. 

The  enzyme  in  a  solution  of  the  gum  from  Japanese  lac  was 
rapidly  destroyed  by  boiling,  but  the  powder,  after  heating  for 
two  hours  at  100°  C.,  was  still  more  active  than  any  of  the  other 
gums  examined.  The  color  with  tincture  of  guaiac  appeared  at 
once  and  in  five  minutes  became  dark  blue.  Another  sample, 
when  heated  for  two  hours  at  120°  C.,  required  ten  minutes  to 
produce  the  same  deep  blue  shade.  A  third  sample,  heated  for 
two  hours  at  140°  C.,  required  ten  minutes  to  produce  any  color, 
but  became  dark  blue  in  thirty  minutes.  A  fourth  sample,  after 
heating  for  two  hours  at  160°  C.,  was  inactive. 


-48 


OXIDATION  PRODUCTS. 

The  gum-enzyme  was  oxidized  by  heating  I  part  of  gum 
with  12  parts  of  nitric  acid  1.15  sp.  gr.  on  a  water  bath  for  one 
hour  then  evaporating  to  2  parts  and  adding  water  2  parts. 
After  24  hours  the  white  crystalline  deposit  was  washed  with 
water  and  alcohol,  and  recrystallized  from  boiling  water.  The 
melting  point  was  the  same  as  for  Mucic  acid  210°  C.  When 
analyzed  : 

0.222  Gm.   gave  0.1085   Gm.   H2O,  0.2844  Gm.,   CO2. 

Calculated  for  Mucic  acid 

C«    H10    Os 
C  ..............  3448%  34-278 

H  .............  4-644  4796 

O   .............  60.926 


100.000 

After  removing  the  mucic  acid  with  hot  water  from  the  first 
crystalline  deposit,  there  remained  a  white  powder  insoluble  in 
hot  water,  alcohol  or  acetic  acid  but  soluble  in  hydrochloric  acid. 
This  was  calcium  oxalate  which  had  been  formed  by  the  union 
of  the  calcium  of  the  gum  with  the  oxalic  acid  formed  by  oxida- 
tion. The  mother  liquor  was  evaporated  to  dryness,  washed  with 
ether,  which  on  evaporation  left  well  defined  crystals  of  tartaric 
acid. 

HYDROLYSIS    OF    LACGUM. 

The  gum-enzyme  was  heated  with  2%  sulphuric  acid  for 
8  hours  and  the  acid  removed  with  barium  hydroxide  and  carbon- 
ate. The  solution  was  evaporated  under  diminished  pressure, 
when  it  formed  a  very  light  yellow  syrup,  non-crystallizable,  non- 
fermentable,  reduced  Fehling's  solution  and  was  dextrorotary. 
Alcohol  dissolved  only  a  small  part  of  it.  The  remainder  could 
be  dissolved  by  using  a  large  quantity  of  hot  alcohol  but  deposited 
on  cooling. 

One  part  of  the  syrup  was  heated  one  hour  with  two  parts 
of  phenylhydrazine,  3  parts  of  sodium  acetate,  and  20  parts  of 
water.  On  cooling  an  abundant  yellow  crystalline  deposit  formed. 
This  was  several  times  recrystallized  from  hot  alcohol,  when  the 
M.  P.  remained  constant  beginning  at  162°  C.,  and  was  complete 
at  164°  without  liberation  of  gas.  The  crystals  were  in  small 
spheroidal  clusters,  which  under  the  microscope  appeared  to  con- 


—  49  — 

sist  of  aggregations  of  needles.  This  corresponds  exactly  with 
the  description  and  melting  point  given  for  phenylsorbinosazone.80 
A  second  crop  of  crystals  was  obtained  by  concentrating  the  moth- 
er liquor.  These  were  somewhat  darker  than  the  first  and  had  a 
M.  P.  of  157°  C.,  but  with  the  production  of  gas.  This  corres- 
ponds with  the  description  given  for  the  osasone  obtained  from 
the  inactive  sorbin. 

Furfurol  was  estimated  from  the  gum  according  to  Tollen's 
method81  and  calculated  as  pentosan  but  the  results  were  not  sat- 
isfactory.   The  method  is  designed  for  the  estimation  of  furfurol 
in  food  products,  and  has  very  little  value  for  scientific  investigation. 
An  analysis  of  the  gum-enzyme  gave  the  following  results : 
I     0.2626  Gm.  gave  0.1424  HSO,  0.402  Gm.  CO2. 
II     0.3464  Gm.  gave  0.1812  H2O,  0.529  Gm.  CO2. 
I     0.35      Gm.  gave  2      cc.  N  at  i8°C.  &  714.7  Mm. 
II     0.452    Gm.  gave  2.3  cc.  N  at  i6°C.  &  713.7  Mm. 

I  II  MEAN 

C     4L742  41-645  -41.693 

H     6.067  S-58i  5.958 

N     0.630  0.587  0.608 

Ash    5.180  5.200  5.190 

O 46.551 

IOO.OOO 

Bertrand82  when  working  with  soluble  oxidizing  ferments 
used  the  gum-enzyme  from  Japanese  lac  under  the  name  "Lac- 
case".  He  reports  that  it  contained  0.44%  of  nitrogen  which  he 
determined  by  heating  with  soda-lime  and  estimating  the  am- 
monia formed  by  titrating  with  decinormal  sulphuric  acid.  From 
this  he  calculated  the  amount  of  enzyme  present  by  assuming 
that  it  has  the  elementary  composition  of  albuminous  substances. 

He  then  gives  the  composition  of  the  gum-enzyme  as : 

Water    74.00% 

Gum   84.95% 

Laccase   2 . 50% 

Ash    S-17% 

From  the  preceding  work  I  think  that  I  am  justified  in  say- 
ing that  what  he  estimated  as  ammonia  was  not  ammonia,  but 
pyrrol. 


80  Vaubel  Quantitative  Bestimmung.    Organ.    Verbindungen  II,  Band 
3,  304. 

81  Lunge.  II  Bel.  460. 

8J  Bull.  Soc.  Chim.  3  series,  51,  p.  259,  1891. 


IMPURITIES   IN    ZINC   DUST 

Before  using  zinc  dust  in  some  experiments  upon  the  gum 
obtained  from  Japanese  lac,  I  wished  to  be  sure  that  it  was  free 
from  nitrogen.  I  therefore  subjected  the  zinc  dust  to  the  follow- 
ing tests,  the  results  of  which  may  be  of  interest  to  those  who 
frequently  use  it  in  connection  with  organic  substances: 

When  heated  with  potassium  hydroxide  it  formed  ammonia. 
When  heated  alone  it  also  gave  off  ammonia.  This  led  to  the 
belief  that  nitrogen  in  some  form  had  been  absorbed  from  the 
atmosphere,  and  might  be  removed  by  heat.  A  small  quantity 
was  therefore  placed  in  a  loosely  covered  crucible,  and  strongly 
heated  for  half  an  hour.  When  cold  it  was  tested  for  nitrogen 
by  heating  with  potassium  hydroxide.  Its  vapors  rapidly  changed 
litmus  paper  from  red  to  blue.  Upon  the  suggestion  of  Profes- 
sor Tschirch  a  sample  was  thoroughly  washed  with  water  acid- 
ulated with  hydrochloric  acid,  but  this  failed  to  completely  re- 
move the  nitrogen. 

As  zinc  dust  is  manufactured  by  heating  zinc  oxide  with  coal, 
it  was  believed  that  part  of  the  nitrogen  might  consist  of  conden- 
sation products  from  the  coal.  Therefore  a  sample  was  placed 
in  a  long  tube  and  percolated  with  ether.  The  ether  when  evap- 
orated left  a  yellow,  non-saponifiable  oil,  with  an  odor  and  fluor- 
escence similar  to  petroleum.  The  oil,  when  heated  with  dry 
potassium  hydroxide,  gave  off  alkaline  vapors,  and  the  zinc  in 
the  percolator  was  still  found  to  contain  nitrogen.  The  greater 
portion  of  the  oil  appeared  to  be  removed  with  the  first  portion 
of  ether,  but  after  continued  percolation  the  ether  left  a  residue 
upon  evaporation,  and  it  was  evident  that  a  much  larger  amount 
of  ether  was  necessary  for  complete  exhaustion  ;  therefore  a  small- 
er sample,  from  a  can  of  zine  dust  which  had  been  in  the  labora- 
tory for  more  than  ten  years,  was  treated  with  ether  in  the  same 
manner,  and  the  powder  tested  from  time  to  time.  After  using 
a  large  amount  of  ether  the  zinc  was  practically  free  from  nitro- 
gen, yet  by  taking  a  large  amount  of  the  zinc  and  heating  with 
potassium  hydroxide  in  a  tube  partially  closed  at  the  top  so  that 
all  of  the  vapors  came  in  contact  with  the  litmus  paper,  the  color 
was  slightly  changed,  thus  showing  a  mere  trace  of  nitrogen. 
This  sample  was  then  allowed  to  stand  in  an  open  flask  for  a  few 
days  when  it  gave  a  decided  ammonia  reaction,  thus  showing  that 
zinc  dust  rapidly  absorbs  nitrogen  from  the  air. 


The  fact  that  only  a  portion  of  the  nitrogen  in  zinc  dust  is 
removed  by  heat  indicates  that  the  nitrogen  is  present  in  more 
than  one  form.  This  theory  is  also  supported  by  the  following 
experiments : 

A  fresh  sample  of  zinc  dust  was  washed  with  water  ,the 
washings  giving  a  decided  ammonia  test.  The  washing  was 
continued  as  long  as  traces  of  nitrogen  could  be  detected  in  the 
washings.  It  was  then  treated  in  the  same  manner  with  very 
dilute  hydrochloric  acid.  By  adding  potassium  hydroxide  in  ex- 
cess to  the  acid  solution  and  allowing  to  stand  a  few  minutes  un- 
til the  precipitate  settled,  decanting  the  clear  solution  and  boil- 
ing, the  vapors  gave  the  odor  of  ammonia  and  rapidly  changed 
litmus  from  red  to  blue.  Washing  with  acid  was  continued  until 
the  washings  no  longer  gave  a  test  for  nitrogen.  The  zinc  was 
then  washed  with  water  until  free  from  acid,  and  rapidly  dried 
in  a  drying  oven,  and  at  once  extracted  with  ether,  the  ether 
evaporated  and  tested  for  nitrogen  as  above.  Nitrogen  was  found 
to  be  present,  though  not  in  as  large  amounts  as  in  the  oil  from 
the  first  sample  examined,  which  was,  however,  directly  treated 
with  ether. 

Three  samples  were  examined :  one  from  a  large  closely 
covered  can  which  had  been  in  use  in  the  laboratory  as  above 
stated ;  another  from  a  glass  bottle  which  had  been  in  the  museum 
about  fifteen  years,  and  a  third  which  was  ordered  by  Professor 
Oesterle  for  these  experiments.  Practically  the  only  difference 
found  in  the  three  samples  was  that  the  oil  from  the  fresh  sample 
was  decidedly  yellow,  while  that  from  the  laboratory  sample  was 
somewhat  lighter,  and  that  from  the  museum  sample  was  colorless. 

Dr.  Victor  Steger  ("Metalldampfe  in  Zinkhiitten,"  Chem- 
ischer  und  Chemischtechnischer  Vortrage)  gives  the  results  of 
several  analyses  of  zinc  dust,  some  of  which  contain  considerable 
insoluble  residue  consisting  principally  of  carbon.  To  determine 
to  what  extent  this  was  present,  a  large  amount  of  zinc  dust 
was  treated  with  hydrochloric  acid.  At  first  the  reaction  was 
rapid,  but  after  a  time  ceased.  The  solution  was  decanted  and 
fresh  acid  added,  but  as  the  reaction  was  very  weak  the  mixture 
was  heated.  Even  then  a  large  amount  remained  undissolved. 
A  few  drops  of  copper  sulphate  solution  were  added  and  digested 
for  several  days,  but  a  large  amount  remained  insoluble.  This 
was  washed  with  water  until  free  from  acid,  dried,  and  per- 


—  52  — 

colated  thoroughly  with  ether,  which  upon  evaporation  left  a  col- 
orless oil.  Upon  removing  the  ether  the  zinc  dissolved  without 
difficulty  in  hydrochloric  acid,  conclusively  proving  that  this 
sample  contained  no  carbon,  and  that  the  insolubility  was  due 
to  the  presence  of  the  oil. 

LAC  POISONING. 

Goertz*3  gives  the  following  description  of  lac  poisoning  in 
which  the  idiosyncrasy  of  the  individual  plays  an  important 
part.  A  few  hours  after  the  poisoning  the  patient  complains  of 
an  unpleasant  tension  of  the  skin,  usually  of  the  face,  head  and 
extremities.  Soon  after  there  forms  an  oedema  of  the  affected 
parts.  Small  red  points  become  visible,  which  look  like  fine  rash. 
These  grow  larger  forming  on  the  points  small  blisters  containing 
a  watery  fluid.  The  parts  of  the  skin  affected  are  restricted  to 
the  head  and  extremities. 

Ishimatsu  states:84  "It  gives  off  a  certain  kind  of  volatile 
acid,  poisonous  in  its  property,  and  some  persons  are  seriously 
attacked  by  it,  producing  great  swellings  on  the  face  especially, 
and  even  the  whole  body  where  the  acid  comes  in  contact.  Dur- 
ing my  examination  in  the  laboratory,  one  day  one  of  the  appara- 
tus-keepers came  in  and  was  violently  attacked  by  it,  producing 
ugly  swellings  all  over  his  face.  He  told  me  at  the  time  that  it 
was  exceedingly  itchy,  and  by  using  solution  of  acetate  of  lead, 
chloride  of  potash  and  carbonate  of  soda,  was  said  to  have  recov- 
ered from  his  suffering  within  a  week." 

"The  poison  that  is  evolved  from  urushi  acts  only  on  certain 
persons.  I  had  to  work  with  it  for  many  days,  yet  never  had  any 
attack  of  the  kind  nor  felt  any  uneasiness  by  it." 

Prof.  J.  J.  Rein86  describes  the  lac  poisoning  as  follows : 

"It  is  a  peculiar,  not  very  painful,  and  not  at  all  fatal,  but 
always  very  disagreeable  disease,  always  attacking  one  new  to 
the  work,  whether  he  be  lac  tapster,  dealer,  or  lacquerer.  It 
appears  in  a  mild  reddening  and  swelling  of  the  back  of  the 
hands,  the  eylids,  ears,  the  region  of  the  navel  and  lower  parts 
of  the  body,  especially  the  scrotum.  In  all  these  parts  great  heat 


83  Ueber  in  Japan  vorkommende   Fish-   und  Lackvergiftungen.     St. 
Petersburger  medicinische  Wochenschrift,  1878,  No.  12. 

•*  Manchester  Literary  and  Philos.  Soc.,  3  Ser.  7,  p.  254. 
85  The  Industries  of  Japan,  p.  349. 


—  53  — 

is  felt  and  violent  itching  and  burning,  causing  many  sleepless 
nights.  In  two  or  three  days  the  crisis  is  reached,  and  the 
swelling  immediately  subsides.  In  severe  cases,  small  festering 
boils  form  also.  This  lacquer  disease  is  not  only  caused  by 
handling  of  the  lac,  but  by  its  evaporation  chiefly,  especially  that 
of  the  sharp  Se-shime,  to  which  I  owe  my  own  illness." 

"The  poison,  however,  is  a  volatile  substance,  and  has  nothing 
to  do  with  the  lac-acid  and  its  higher  oxidation,  as  Korschelt 
believed.  If  the  poisonous  property  disappears  in  the  drying  of 
the  plant,  this  amounts  to  nothing  save  that  the  volatile  poison 
fully  escapes  in  this  manner.  A  considerable  part  of  it  is  driven 
off  in  the  preparation  of  the  several  kinds  of  lacquer,  and  by 
stirring  in  open  vessels.  For  this  reason,  the  lacquers  mixed 
with  colors  are  regarded  far  less  dangerous  than  the  raw  lac 
and  its  direct  derivatives." 

ForneP  compares  the  above  symptoms  with  those  of  poison- 
ing by  poison  ivy  and  anarcardium,  as  well  as  the  cases  of  poison- 
ing of  the  laborers  in  the  vanilla  depot  of  Bordeaux"  where 
nearly  all,  even  from  the  first  day,  experienced  strong  itching 
accompanied  with  a  burning  sensation,  especially  on  the  face  and 
hands.  He  finds  a  remarkable  similarity  in  all  of  these  cases  and 
believes  that  he  is  justified  in  assuming  that  all  are  produced  by 
cardol.  He  also  states  that  anacardium  is  used  in  the  preparation 
of  Japanese  lac,  but  I  have  been  unable  to  find  confirmation  of 
this  fact. 

Dr.  Andreas88  reports  the  case  of  a  gardener  in  the  botanical 
garden  at  Vienna,  who  was  poisoned  while  collecting  and  trans- 
planting Rhus  vernicifera.  On  the  same  day  the  face  became 
red,  the  skin  inflamed,  the  eyelids,  nose  and  cheeks  swollen.  The 
reddening  extended  to  the  neck,  breast,  hands  and  forearms. 
The  genitals  were  also  red  and  swollen.  By  dusting  with  starch 
the  inflammation  disappeared  in  fourteen  days. 

When  opening  the  cans  care  must  be  exercised  to  prevent  the 
vapors  accumulated  in  the  top  of  the  can  from  coming  in  contact 
with  the  face  or  hands,  as  the  poisonous  part  of  the  lac  is  volatile 
and  may  be  removed  by  heating  or  by  distillation.  Yoshida  also 
states  that  the  lac  contains  a  volatile  poison  which  is  dissolved 
with  the  urushic  acid  by  alcohol  but  is  almost  completely  driven 


Archiv.  fur  Dermatologie  und  Syphilis,  L,X,  p.  249. 
Revue  d'Hygiene,  Paris,  1883,  p.  718. 
Therap.  Monotshifte,  1903,  p.  165. 


—  54  — 

off  by  drying  the  acid  at  105°  to  110°  C.  Bertrand89  says  that 
the  lac  must  be  handled  with  the  greatest  precaution  because  the 
least  traces  in  the  state  of  vapor  produce  on  the  face,  hands  and 
arms  an  intense  rubrefaction  accompanied  by  intense  itching,  and 
adds  that  these  malicious  properties  make  the  study  of  lac  very 
unpleasant,  and  he  was  obliged  to  interrupt  his  studies  on  account 
of  individual  sensibilities. 

With  these  statements  before  me  it  was  not  without  mis- 
givings that  I  undertook  the  study  of  lac,  and  these  were  not 
allayed  by  my  first  experience.  The  first  sample  received  was 
in  a  glass  can  with  metal  top  which  had  become  sealed  by  the 
lac,  and  was  difficult  to  remove,  but  when  finally  started  was 
accompanied  by  a  slight  sound  of  escaping  gas.  In  about  thirty- 
six  hours  an  inflamed  spot  about  2  cm.  by  5  cm.  appeared  on  my 
wrist ;  it  itched  intensely  for  about  a  week  and  then  disappeared. 
Laboring  under  the  supposition  that  I  was  dealing  with  a  volatile 
poison,  I  was  extremely  cautious  not  to  come  in  contact  with  the 
vapors  in  any  form,  but  supposed  that  I  was  practically  safe  after 
the  alcohol  had  been  distilled  and  the  residue  had  been  heated 
for  some  time.  While  shaking  out  an  ether  solution  of  the 
alcoholic  residue  with  sodium  carbonate  solution,  it  was  difficult 
to  keep  the  hands  entirely  free  from  the  solution  and  no  especial 
pains  were  taken  to  remove  it  except  to  carefully  wash  with  soap 
and  water.  However,  after  working  some  time  with  it  my  face 
began  to  swell  and  continued  until  my  eyes  were  nearly  closed. 
It  extended  over  hands,  arms  and  limbs  to  the  knees ;  the  desire 
to  scratch  was  very  great  so  that  it  was  almost  impossible  to 
sleep.  This  was  also  true  of  the  face  and  ears  to  some  extent, 
but  here  the  sensation  was  more  that  of  burning.  After  about  a 
week  the  face  became  normal  and  I  was  able  to  resume  my  work 
but  the  limbs  continued  to  itch  and  remained  covered  with  a  fine 
rash.  After  several  weeks  I  became  convinced  that  the  underwear 
had  absorbed  some  of  the  poison  and  though  frequently  washed 
still  retained  it.  Soft  gauze  underwear  was  then  worn  next  the 
skin,  when  the  flesh  soon  became  normal. 

Various  remedies  were  tried,  such  as,  ointment  of  zinc 
oxide ;  a  mixture  of  oxide  of  zinc,  bismuth  subnitrate,  starch  and 
solution  lead  subacetate,  tincture  of  iodine  with  glycerin ;  solu- 
tion of  potassium  permanganate ;  solution  of  oxalic  acid.  None 


Annales  de  Chimie  et  de  Physique,  Series  XII,   1897. 


—  55  — 

of  these  seemed  to  give  anything  more  than  a  temporary  relief. 
The  best  results  were  obtained  by  rubbing  the  surface  with  a  little- 
petrolatum  and  then  scraping  it  off  with  a  knife  and  washing  the 
surface  with  a  weak  solution  of  sodium  hydroxide  or  carbonate. 
The  burning  on  the  face  was  relieved  by  keeping  it  moistened 
with  a  saturated  solution  of  boric  acid.  > 

Dr.  Jadassohn,  Professor  of  Skin  Diseases  in  the  University 
of  Bern,  stated  that  the  above  symptoms  did  not  prove  that  the 
poisonous  principle  was  volatile,  and  kindly  volunteered  to  make 
the  physiological  tests  for  me  in  order  to  determine  whether  the 
poisonous  principle  is  volatile  or  not.  He  found  that  the  rabbit 
was  very  sensitive  to  the  poison.  The  method  of  testing  was  to 
rub  a  small  quantity  of  the  substance  on  the  inside  of  the  ear  for 
2  or  3  minutes.  If  poisonous,  inflammation  appeared  in  from 
i  to  5  days  and  the  surface  soon  became  covered  with  watery 
blisters  followed,  in  severe  cases  by  necrosis  of  the  superficial 
layers  of  the  skin.  This  condition  lasted  about  14  days  when  it 
gradually  disappeared. 

The  following  are  the  most  important  results  obtained  from 

the  tests : 

1.  Sterilized  lac,  prepared  by  suspending  a  tube  of  the  lac  in 
boiling  water  for  half  an  hour,  was  poisonous. 

2.  An  alcoholic  solution  of  the  lac  was  distilled  and  the  dis- 
tillate tested  but  was  not  poisonous. 

3.  After  the  alcohol  was  removed,  the  distillation  was  con- 
tinued when  a  small  quantity  of  aqueous  distillate  was  obtained, 
but  this  was  also  inactive. 

4.  The  residue  in  the  retort  was  extremely  poisonous. 

5.  A  fresh  can  of  lac  was  thoroughly  cooled  to  prevent  the 
escape  of  gas  while  opening,  two  small  openings  made,  and  tubes 
introduced.     A  small  quantity  of  absorbent  cotton  was  placed 
in  the  tube,  used  for  the  exit  of  vapor,  to  prevent  particles  of  the 
fluid  from  being  forced  through.     The  vapor  was  then  slowly 
forced  out  of  the  can  upon  the  ear  of  a  rabbit.     Part  of  the  ear 
had  previously  been  moistened.    The  vapor  was  entirely  without 
action.     Since  then  I  have  worked  over  the  lac,  while  evaporating 
it  under  all  conditions  without  the  slightest  inconvenience. 

6.  The  alcoholic  residue  was  later  separated  into  two  parts, 
one  soluble  and  the  other  insoluble  in  benzin.     The  first  was 


-56- 

poisonous  and  the  second  non-poisonous.  A  thin  layer  of  the 
first  was  left  in  an  open  crystallizing  jar  for  four  months  when 
it  was  found  to  be  still  poisonous. 

Another  sample  of  five  grams  was  left  in  an  open  vial  on  a 
laboratory  shelf  for  ten  months,  including  the  hot  summer  months. 
This  was  then  tested  on  my  arm  and  was  found  to  be  still  active. 
These  facts  are  sufficient  to  prove  that  the  poisonous  principle 
is  non-volatile.  Doubtless  the  cases  of  poisoning  that  have  oc- 
curred from  opening  retainers  have  been  due  to  minute  particles 
of  the  lac  being  forced  out  with  the  vapor. 

The  poison  is  extremely  active  even  in  minute  quantities  and, 
as  it  forms  a  part  of  the  resinous  body,  it  is  very  difficult  to  remove 
from  the  skin  or  clothing.  Washing  with  soap  and  water  is  not 
sufficient  to  insure  its  removal.  If  the  hands  after  contact  with 
the  lac  are  thoroughly  washed  with  soap  and  water  until  they 
are  to  all  appearances  clean,  and  then  wet  with  a  solution  of  caus- 
tic alkali,  black  spots  will  appear  wherever  the  lac  has  been  in 
contact.  A  mixture  of  powdered  soap,  pumice  stone  and  sodium 
carbonate  gives  the  best  result.  However,  to  insure  safety 
I  have  usually  followed  this  with  soap  and  sand.  The  poison 
seems  to  have  little  or  no  effect  upon  the  thick  skin  on  the  inside 
of  the  hand,  but,  to  prevent  its  transmission  to  other  parts,  it  should 
be  removed  as  soon  as  possible.  For  example,  by  accident  some 
of  the  benzin  solution  was  thrown  into  one  eye  and  over  one 
hand.  The  eye  was  thoroughly  washed  with  benzin  and  alcohol, 
but  in  my  anxiety  for  the  eye,  the  hand  was  forgotten  for  twen- 
ty or  thirty  minutes,  when  it  was  thoroughly  washed  with  ben- 
zin and  alcohol  followed  by  soap  and  sand.  The  eye  escaped 
without  further  inconvenience  than  that  caused  by  the  benzin, 
but  in  thirty-six  hours  the  surface  of  the  hand  became  slightly 
swollen,  itched  considerably  for  a  week  and  then  appeared  to 
be  covered  with  a  thin  dry  scale,  which  finally  disappeared.  Since 
then  I  have  tested  different  parts  of  the  substance  to  determine 
whether  or  not  they  were  poisonous,  by  cutting  a  hole  6  mm.  in 
diameter  in  a  piece  of  gum  paper,  pasting  this  on  the  arm  and 
applying  the  substance  to  the  opening.  In  from  thirty  minutes 
to  one  hour  the  paper  was  removed  and  the  spot  washed  with 
ether  or  benzin.  When  the  substance  was  poisonous  the  spot 
became  red  and  began  to  itch  within  30  hours.  From  three  to 
five  vescicles  usually  appeared.  The  itching  was  not  intense, 


—  57  — 

usually  lasting  only  a  few  minutes  at  a  time.  A  dry  scale  formed 
over  the  surface  and  remained  for  several  weeks  after  all  irrita- 
tion ceased. 

In  no  case  did  the  poisonous  action  extend  beyond  the  sur- 
face to  which  it  was  applied,  thus  proving  that  the  action  is  en- 
tirely local.  If  the  surface,  which  has  been  in  contact  with  the 
poison,  is  not  thoroughly  washed  with  some  solvent  like  alcohol, 
benzin,  ether  or  kerosene,  the  poison  will  be  transmitted  to  other 
parts  of  the  body  As  all  kinds  of  fats  and  oils  are  solvents  for  the 
poison  they  should  not  be  used  as  remedies.  Should  pustules  form, 
the  surface  should  be  frequently  washed  to  prevent  the  serum  from 
being  conveyed  to  other  parts,  as  it  is  quite  possible  that  it  may 
be  active.  Experiments  to  determine  this  fact  will  be  conducted 
in  the  near  future. 

The  poison  has  not  at  the  present  time  been  isolated  in  a 
pure  condition. 

Dr.  Jadassohn  and  his  assistants,  Drs,  Winckler  and  Schulz, 
made  26  tests  with  parts  of  the  lac  obtained  under  different  con- 
ditions. 

Only  that  portion  which  is  completely  soluble  in  benzin  is 
poisonous,  and  this,  we  have  previously  seen,  was  separated 
by  shaking  out  the  benzin  solution  with  alcohol,  into  two  parts, 
one  soluble  in  alcohol  and  poisonous,  the  other  insoluble  in  alco- 
hol but  soluble  in  benzin  and  non-poisonous.  I  have  elsewhere 
stated  that  by  fractional  precipitation  with  lead  acetate  a  partial 
separation  of  the  poison  was  obtained,  but  that  I  did  not  consider 
it  a  practical  method. 

After  the  above  experiments  with  the  poison  were  made  I 
received  from  Dr.  F.  Pfaff  a  reprint  of  his  article  "On  the  Ac- 
tive Principle  of  Rhus  Toxicodendron  and  Rhus  Venenata."90  As 
the  poisonous  action  of  these  plants  is  practically  identical  with 
that  of  Rhus  vernicifera,  his  work  is  of  special  interest  in  this 
connection.  He  has  conclusively  proved  that  the  poisonous  prin- 
ciple of  poison  ivy  is  non-volatile,  thus>  shatter'"^  the  false  idea 
that  has  existed  for  so  many  years.  He  claims  to  have  separated 
the  poisonous  principle  in  a  pure  form  by  fractional  precipitation 
with  lead  acetate  as  an  oil.  Dr.  Pfaff  gives  the  composition  of 
his  lead  compound  as  C21H30O4Pb6  and  proposes  the  name  "Tox- 


10  The  Journal   of  Experimental   Medicine,  Vol.   II,   No.   2,   p.    181, 
1897. 


-58- 

icodendrol"  as  the  name  of  the  poisonous  principle.  The  poison- 
ous principle  of  Japanese  lac  is  so  intimately  associated  with  the 
resin  of  the  lac  that  I  have  not  considered  the  method  of  fraction- 
al precipitation  to  be  a  complete  separation.  Preceding  investiga- 
tions indicate  that  the  poisonous  principles  of  these  plants  are 
identical  but  further  investigation  is  necessary  before  this  can  be 
accepted  as  conclusive.  I  hope  during  the  coming  year  to  be  able 
to  separate  the  poison  from  both  these  plants  and  to  determine 
their  relation. 


The  present  researches  in  Japanese  Lac  were  undertaken  in 
the  Laboratory  of  the  Pharmaceutical  Institute  of  Bern  under 
the  guidance  of  my  most  highly  esteemed  director,  Professor  A. 
Tschirch.  To  him  and  also  to  Professor  Oesterle  I  desire  to  ex- 
press my  warmest  and  sincerest  thanks  for  the  inspiration  and 
the  friendly  interest  and  advice  which  has  ever  been  so  freely 
and  so  kindly  given. 

I  am  also  thankful  to  Dr.  Jadassohn  and  his  assistants,  Drs. 
Winckler  and  Schulz  for  the  physiological  tests  which  they  so 
kindly  made. 

The  lac  for  this  investigation  was  kindly  presented  by  fores- 
ter Shirasawa  of  Tokio,  Japan,  and  the  Rhus  Company,  Frank- 
fort, Germany.  To  them  I  extend  sincere  thanks. 


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